Antibodies

ABSTRACT

Antibodies that bind human β-amyloid peptide, methods of treating diseases or disorders characterised by elevated β-amyloid levels or β-amyloid deposits with said antibodies, pharmaceutical compositions comprising said antibodies and methods of manufacture.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/294,438, filed Sep. 25, 2008 which claims benefit to InternationalApplication No. PCT/EP2007/052928, filed Mar. 27, 2007, which claimsbenefit to U.S. Provisional Application 60/787,588 filed Mar. 30, 2006.The entire teachings of the above applications are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind β-amyloid peptideand in particular human β-amyloid peptide. The present invention alsoconcerns methods of treating diseases or disorders characterised byelevated β-amyloid levels or β-amyloid deposits, particularlyAlzheimer's disease, with said antibodies, pharmaceutical compositionscomprising said antibodies and methods of manufacture. Other aspects ofthe present invention will be apparent from the description below.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is the most common cause of age-relatedcognitive decline, affecting greater than 12 million individualsworldwide (Citron M (2002) Nat. Neurosci 5, Suppl 1055-1057). Theearliest stages of the disease are characterized by a progressive lossof memory with associated cognitive decline and language and behaviouraldeficits. In the later stages of the disease, patients develop globalamnesia and have greatly reduced motor function. Death typically occurs9 years following diagnosis and is often associated with otherconditions, typically pneumonia (Davis K. L. and Samules S.C. (1998) inPharmacological Management of Neurological and Psychiatric Disorders edsEnna S. J. and Coyle J. T. (McGraw-Hill, New York pp 267-316)). Currenttherapies represent symptomatic approaches, focussing on alleviating thecognitive impairment and ameliorating the behavioural symptomsassociated with the progressing disease aetiology. In practice thesetreatments provide only a short lived cognitive benefit with the levelof cognitive impairment reported only to last up to 2 years. Thepotential for a disease-modifying therapy that slows and possibly haltsthe progression of the disease is enormous. Such approaches wouldprovide radical and sustained improvements to the quality of life ofpatients and importantly their carers as well as reducing the hugeoverall healthcare costs of this disease.

Clinical diagnosis of Alzheimer's disease is based upon a combination ofphysical and mental tests which lead to a diagnosis of possible orprobable Alzheimer's disease. At post mortem the disease is confirmed bywell characterised neurological hallmarks in the brain, which includethe deposition of Aβ in parenchymal plaques and cerebral vessels,intraneuronal formation of neurofibrillary tangles, synaptic loss andloss of neuronal subpopulations in specific brain regions (Terry, R D(1991) J Neural Trans Suppl 53: 141-145).

A plethora of genetic, histological and functional evidence suggeststhat the β-amyloid peptide (Aβ) is key to the progression of Alzheimer'sdisease (Selkoe, D. J. (2001) Physiological Reviews 81: 741-766).

Aβ is known to be produced through the cleavage of the beta amyloidprecursor protein (also known as APP) by an aspartyl protease enzymeknown as BACE1 (also known as β-secretase, Asp2 or Memapsin-2) (DeStrooper, B. and Konig, G. (1999) Nature 402: 471-472). In addition tothe parenchymal and vascular deposition, soluble oligomeric forms of Aβhave been postulated to contribute to the onset of AD and they mayaffect neuronal function initially by impairing synaptic function(Lambert et. al. (1998) Proceedings of the National Academy of Science,U.S.A. 95: 6448-6453). Although insoluble amyloid plaques are foundearly in AD and in MC1, the levels of soluble Aβ aggregates (referred toas oligomers or Aβ-derived diffusible ligands (ADDLs) are also increasedin these individuals, and soluble Aβ levels correlate better withneurofibrillary degeneration, and the loss of synaptic markers than doamyloid plaques (Naslund et. al. (2000) J Am Med Assoc 283: 1571-1577,Younkin, S. (2001) Nat. Med. 1: 8-19). The highly amyloidogenic Aβ42 andaminoterminally truncated forms Mx-42 are the predominant species of Aβfound in both diffuse and senile plaques (Iwatsubo, T (1994) Neuron.13:45-53, Gravina, S A (1995) J. Biol. Chem. 270:7013-7016) The relativelevels of Aβ42 appear to be the key regulator of Aβ aggregation intoamyloid plaques, indeed Aβ42 has been shown to aggregate more readilythat other Aβ forms in vitro (Jarrett, J T (1993) Biochemistry.32:4693-4697) and as such Aβ42 has been implicated as the initiatingmolecule in the pathogenesis of AD (Younkin S G, (1998) J. Physiol.(Paris). 92:289-292). Although Aβ42 is a minor product of APPmetabolism, small shifts in it's production are associated with largeeffects on Aβ deposition therefore it has been postulated that reductionof Aβ42 alone may be an effective way of treating AD (Younkin S G,(1998) J. Physiol. (Paris). 92:289-292) In support of this, mutations inthe amyloid precursor protein (APP) and presenilin genes have beenreported to predominantly increase the relative levels of Aβ42 andtherefore shortening the time to onset of Alzheimer's disease (AD)(Selkoe D. J., Podlisny M. B. (2002) Annu. Rev. Genomics Hum. Gemet.3:67-99). It should be noted however, that the rate of deposition isalso dependant on catabolism and Aβ clearance.

Animal models of amyloid deposition have been generated byoverexpressing mutant human transgenes in mice. Mice overexpressingsingle human APP transgenes typically develop cerebral plaque-likeβ-amyloid deposits from 12 months of age (Games D. et al., (1995) Nature373: 523-527; Hsiao K. et al., (1996) Science 274: 99-102)), while micecarrying both mutant human APP and presenilin-1 (PS-1) transgenestypically develop cerebral plaque-like β-amyloid deposits as early as 2months of age (Kurt M. A. et al., (2001) Exp. Neurol. 171: 59-71;McGowan E. et al., (1999) Neurolbiol. Dis. 6: 231-244.

It has become increasingly apparent that the transport of exogenous Aβbetween the central nervous system (CNS) and plasma plays a role in theregulation of brain amyloid levels (Shibata, et al (2000) J Clin Invest106: 1489-1499), with CSF Aβ being rapidly transported from CSF toplasma. Therefore active vaccination with Aβ peptides or passiveadministration of specific Aβ antibodies rapidly binds peripheral Aβaltering the dynamic equilibrium between the plasma, CSF and ultimatelythe CNS. Indeed there are now a plethora of studies demonstrated boththese approaches can lower Aβ levels, reduce Aβ pathology and providecognitive benefit in various transgenic models of amyloidosis. Limitedstudies have also been conducted in higher species. Caribbean vervetmonkeys (16-10 years old) were immunised with Aβ peptide over 10 months.Aβ40 levels were elevated 2-5 fold in the plasma which peaked at 251dwhile the CSF levels of Aβ40 and Aβ42 were significantly decreased by100d and returned towards baseline thereafter. This reduction in CSF wasaccompanied by a significant reduction in plaque burden (Lemere, Calif.(2004) Am J Pathology 165: 283-297). Similar increases in plasma Aβlevels were also detected following immunisation of aged (15-20 yearold) Rhesus Monkeys (Gandy, S (2004) Alzheimer Dis Assoc Disord 18:44:46.

The first immune therapy targeting brain amyloid was Elan/Wyeth'sAN-1792, an active vaccine. This treatment was terminated following thedevelopment of clinical signs consistent with meningoencephalitis.Subgroup analyses suggested that treatment slowed the decline ofcognitive function (Nature Clin Pract Neurol (2005) 1:84-85).Post-mortem analysis of patients also showed evidence ofplaque-clearance (Gilman S. et al, (2005) Neurology 64 (9) 1553-1562).Bapineuzumab (AAB-001, Elan/Wyeth), a passive MAb therapy has been shownto significantly improve cognition scores in a small phase I safetystudy.

Other diseases or disorders characterised by elevated β-amyloid levelsor β-amyloid deposits include mild cognitive impairment (MC1, Blasko I(2006) Neurobiology of aging “Conversion from cognitive health to mildcognitive impairment and Alzheimer's disease: Prediction by plasmaamyloid beta 42, medial temporal lobe atrophy and homocysteine” inpress, e-published 19 Oct. 2006), hereditary cerebral haemorrhage withβ-amyloidosis of the Dutch type, cerebral β-amyloid angiopathy andvarious types of degenerative dementias, such as those associated withParkinson's disease, progressive supranuclear palsy, cortical basaldegeneration and diffuse Lewis body type of Alzheimer's disease(Mollenhauer B (2007) J Neural Transm e-published 23 Feb. 2007, vanOijen, M Lancet Neurol. 2006 5:655-60) and Down syndrome (Mehta, PD(2007) J Neurol Sci. 254:22-7).

SUMMARY OF THE INVENTION

In an embodiment of the present invention there is provided atherapeutic antibody which is an antibody or antigen binding fragmentand/or derivative thereof which binds β-amyloid peptide 1-12 (SEQ IDNo:15) with equilibrium constant KD less than 100 μM but does not bindto β-amyloid peptide 2-13 (SEQ ID No:44), both determinations being madein a surface plasmon resonance assay utilising peptide captured onstreptavidin chip.

In another embodiment of the present invention there is provided atherapeutic antibody which is an antibody or antigen binding fragmentand/or derivative thereof which binds β-amyloid peptide 1-12 (SEQ IDNo:15) with equilibrium constant KD less than 100 μM and has anequilibrium constant KD for binding to β-amyloid peptide 2-13 (SEQ IDNo:44) which is 1000-fold greater than that for peptide 1-12 (SEQ IDNo:15), both determinations being made in a surface plasmon resonanceassay utilising peptide captured on streptavidin chip.

In another embodiment of the present invention there is provided atherapeutic antibody which is an antibody or antigen binding fragmentand/or derivative thereof which binds β-amyloid peptide 1-12 (SEQ IDNo:15) with equilibrium constant KD less than 100 μM and has anequilibrium constant KD for binding to β-amyloid peptide 2-13 (SEQ IDNo:44) which is 10.000-fold greater than that for peptide 1-12 (SEQ IDNo:15), both determinations being made in a surface plasmon resonanceassay utilising peptide captured on streptavidin chip.

In one aspect the surface plasmon resonance assay utilising peptidecaptured on streptavidin chip is the Surface Plasmon Resonance assaydescribed in the Example below. In another aspect the surface plasmonresonance assay utilising peptide captured on streptavidin chip is theMethod A(i) described under SPR Biacore™ Analysis below.

In an alternative embodiment of the present invention there is provideda therapeutic antibody which is an antibody or antigen binding fragmentand/or derivative thereof which binds β-amyloid peptide 1-40 withequilibrium constant KD less than 10 nM but does not bind to β-amyloidpeptide 2-13 (SEQ ID No:44), both determinations being made in thesurface plasmon resonance assay described in Method B of the Examplesbelow.

In another alternative embodiment of the present invention there isprovided a therapeutic antibody which is an antibody or antigen bindingfragment and/or derivative thereof which binds β-amyloid peptide 1-40with equilibrium constant KD less than 10 nM and has an equilibriumconstant KD for binding to β-amyloid peptide 2-13 (SEQ ID No:44) whichis 1000-fold greater than that for peptide 1-12 (SEQ ID No:15), bothdeterminations being made in the surface plasmon resonance assaydescribed in Method B of the Examples below.

In another alternative embodiment of the present invention there isprovided a therapeutic antibody which is an antibody or antigen bindingfragment and/or derivative thereof which binds β-amyloid peptide 1-40with equilibrium constant KD less than 10 nM and has an equilibriumconstant KD for binding to β-amyloid peptide 2-13 (SEQ ID No:44) whichis 10.000-fold greater than that for peptide 1-12 (SEQ ID No:15), bothdeterminations being made in the surface plasmon resonance assaydescribed in Method B of the Examples below.

In an embodiment of the present invention there is provided atherapeutic antibody which is an antibody or antigen binding fragmentand/or derivative thereof which binds β-amyloid peptide and whichcomprises the following CDRs:

CDRH1: (SEQ ID No: 1) DNGMA CDRH2: (SEQ ID No: 2) FISNLAYSIDYADTVTGCDRH3: (SEQ ID No: 3) GTWFAYwithin a human heavy chain variable region originating from the VH3 genefamily and:

CDRL1: (SEQ ID No: 4) RVSQSLLHSNGYTYLH CDRL2: (SEQ ID No: 5) KVSNRFSCDRL3: (SEQ ID No: 6) SQTRHVPYTwithin a human light chain variable region originating from the aminoacid sequence disclosed in GenPept entry CAA51135 (SEQ ID No:24).

Throughout this specification, the terms “CDR”, “CDRL1”, “CDRL2”,“CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” follow the Kabat numbering system asset forth in Kabat et al; Sequences of proteins of ImmunologicalInterest NIH, 1987. Therefore the following defines the CDRs accordingto the invention:

CDR: Residues CDRH1: 31-35B CDRH2: 50-65 CDRH3: 95-102 CDRL1: 24-34CDRL2: 50-56 CDRL3: 89-97

The VH3 gene family and related immunoglobulin gene nomenclature isdescribed in Matsuda et al (Journal of Experimental Medicine,188:2151-2162, 1998) and Lefranc & Lefranc (The ImmunoglobulinFactsbook. 2001. Academic Press: London).

In a particular embodiment, the human heavy chain variable regionoriginates from:

-   -   A V gene selected from the following subset of VH3 family        members: VH3-48, VH3-21, VH3-11, VH3-7, VH3-13, VH3-74, VH3-64,        VH3-23, VH3-38, VH3-53, VH3-66, VH3-20, VH3-9 & VH3-43    -   A V gene selected from the following subset of VH3 family        members: VH3-48, VH3-21 & VH3-11    -   The VH3-48 gene        or an allele thereof.

The sequence in Genbank entry M99675 is an allele of the VH3-48 gene.M99675 is a Genbank nucleotide sequence of a genomic piece of DNAincluding the two exons that constitute the human heavy chain geneVH3-48 (SEQ ID No:22) and encode the variable region amino acid sequencegiven in SEQ ID No:21. In a particular aspect the human acceptor heavychain framework is derived from M99675.

In order to construct a complete V-region a framework 4 has to be addedto the germline encoded V-gene M99675. Suitable framework 4 sequencesinclude that encoded by the human JH4 minigene (Kabat):

(SEQ ID No: 23) YFDYWGQGTLVTVSSof which the initial four residues fall within the CDR3 region which isreplaced by the incoming CDR from the donor antibody.

The skilled person appreciates that a germline V gene and a J gene donot include coding sequence for the entirety of heavy chain CDR3.However, in the antibodies of the invention, the CDR3 sequence isprovided by the donor immunoglobulin. Accordingly, the combination of aVH gene such as VH3-48, a JH minigene such as JH4, and a set of heavychain CDRs, such as SEQ ID No:1, SEQ ID No:2 and SEQ ID No:3 (assembledin a manner so as to mimic a mature, fully rearranged heavy chainvariable region) is sufficient to define a heavy chain variable regionof the invention such as that represented in SEQ ID No:26, 28, 30.

The variable region encoded by Genpept ID CAA51134 has the amino acidsequence given in SEQ ID No:24.

The light chain variable region framework sequence known by the GenPeptID CAA51134 is the deduced amino acid sequence of a fully rearrangedlight chain variable region and is identical to another amino acidsequence with the same frameworks in the database: Genpept accessionnumber S40356, and is described in Klein, R., et al., Eur. J. Immunol.23 (12), 3248-3262 (1993). The DNA coding sequence for CAA51134,accessible as Genbank Accession No X72467, is given as SEQ ID No: 25.

In a particular aspect of the invention the human acceptor heavy chainframework is derived from M99675 and the JH4 minigene and the humanacceptor light chain framework is derived from CAA51135, optionallycontaining one or more, such as one to four, more particularly one tothree, substitutions of amino acid residues based on the correspondingresidues found in the donor V_(H) domain having the sequence: SEQ IDNo:17 and V_(L) domain having the sequence: SEQ ID No: 19 that maintainall or substantially all of the binding affinity of the donor antibodyfor β-amyloid peptide.

By ‘substantially all of the binding affinity’ is meant that thetherapeutic antibody has at most a ten-fold reduction in bindingaffinity compared to the donor antibody.

In a more particular aspect the human acceptor heavy chain frameworkderived from M99675 and JH4 has one to four amino acid residuesubstitutions selected from positions 24, 48, 93 and/or 94 (Kabatnumbering).

In a more particular aspect of the invention the human acceptor heavychain framework derived from M99675 and JH4 comprises the followingresidues (or a conservative substitute thereof):

Position Residue (i) 93 V 94 S or (ii) 24 V 93 V 94 S or (iii) 48 I 93 V94 S

In one embodiment of the invention there is provided a therapeuticantibody comprising a V_(H) chain having the sequence set forth in SEQID No:26, 28 or 30 and a V_(L) domain having the sequence set forth inSEQ ID No:32.

In another embodiment of the invention there is provided a therapeuticantibody, which antibody comprises a heavy chain having the sequence setforth in SEQ ID No:34, 36 or 38 and a light chain having the sequenceset forth in SEQ ID No:40.

In another embodiment of the invention there is provided apharmaceutical composition comprising a therapeutic antibody accordingto the invention.

In a further embodiment of the invention there is provided a method oftreating a human patient afflicted with a β-amyloid peptide-relateddisease which method comprises the step of administering to said patienta therapeutically effective amount of a therapeutic antibody accordingto the invention.

Use of a therapeutic antibody according to the invention in themanufacture of a medicament for the treatment of a β-amyloidpeptide-related disease is also provided.

In another embodiment of the invention there is provided a process forthe manufacture of a therapeutic antibody according to the invention,which process comprises expressing polynucleotide encoding the antibodyin a host cell.

In another embodiment of the invention there is provided apolynucleotide encoding a therapeutic antibody heavy chain comprising aV_(H) chain having the sequence set forth in SEQ ID No:26, 28 or 30.

In another embodiment of the invention there is provided apolynucleotide encoding a therapeutic antibody light chain comprising aV_(L) domain having the sequence set forth in SEQ ID No:32.

In another embodiment of the invention there is provided apolynucleotide encoding a therapeutic antibody heavy chain having thesequence set forth in SEQ ID No:34, 36 or 38.

In another embodiment of the invention there is provided apolynucleotide encoding a therapeutic antibody light chain having thesequence set forth in SEQ ID No:40.

In a more particular embodiment of the invention there is provided apolynucleotide encoding a therapeutic antibody heavy chain, whichpolynucleotide comprises the sequence set forth in SEQ ID No:35, 37, 39or 42.

In another more particular embodiment of the invention there is provideda polynucleotide encoding a therapeutic antibody light chain, whichpolynucleotide comprises the sequence set forth in SEQ ID No:41 or 43.

In a particular embodiment the therapeutic antibody which is an antibodyor fragment and/or derivative thereof essentially lacks the functions ofa) activation of complement by the classical pathway; and b) mediatingantibody-dependent cellular cytotoxicity.

In another embodiment of the invention there is provided an antibody ora fragment thereof comprising a V_(H) domain having the sequence: SEQ IDNo:17 and a V_(L) domain having the sequence: SEQ ID No: 19.

In another embodiment of the invention there is provided apolynucleotide encoding an antibody heavy chain or a fragment thereofcomprising a V_(H) domain having the sequence SEQ ID No:17, inparticular the polynucleotide of SEQ ID No:18.

In another embodiment of the invention there is provided apolynucleotide encoding an antibody light chain or a fragment thereofcomprising a V_(L) domain having the sequence SEQ ID No: 19, inparticular the polynucleotide of SEQ ID No:20.

DETAILED DESCRIPTION OF THE INVENTION 1. Antibody Structures 1.1 IntactAntibodies

Intact antibodies are usually heteromultimeric glycoproteins comprisingat least two heavy and two light chains. Aside from IgM, intactantibodies are heterotetrameric glycoproteins of approximately 150 KDa,composed of two identical light (L) chains and two identical heavy (H)chains. Typically, each light chain is linked to a heavy chain by onecovalent disulfide bond while the number of disulfide linkages betweenthe heavy chains of different immunoglobulin isotypes varies. Each heavyand light chain also has intrachain disulfide bridges. Each heavy chainhas at one end a variable domain (V_(H)) followed by a number ofconstant regions. Each light chain has a variable domain (V_(L)) and aconstant region at its other end; the constant region of the light chainis aligned with the first constant region of the heavy chain and thelight chain variable domain is aligned with the variable domain of theheavy chain. The light chains of antibodies from most vertebrate speciescan be assigned to one of two types called Kappa and Lambda based on theamino acid sequence of the constant region. Depending on the amino acidsequence of the constant region of their heavy chains, human antibodiescan be assigned to five different classes, IgA, IgD, IgE, IgG and IgM.IgG and IgA can be further subdivided into subclasses, IgG1, IgG2, IgG3and IgG4; and IgA1 and IgA2. Species variants exist with mouse and rathaving at least IgG2a, IgG2b. The variable domain of the antibodyconfers binding specificity upon the antibody with certain regionsdisplaying particular variability called complementarity determiningregions (CDRs). The more conserved portions of the variable region arecalled framework regions (FR). The variable domains of intact heavy andlight chains each comprise four FR connected by three CDRs. The CDRs ineach chain are held together in close proximity by the FR regions andwith the CDRs from the other chain contribute to the formation of theantigen binding site of antibodies. The constant regions are notdirectly involved in the binding of the antibody to the antigen butexhibit various effector functions such as participation in antibodydependent cell-mediated cytotoxicity (ADCC), phagocytosis via binding toFcγ receptor, half-life/clearance rate via neonatal Fc receptor (FcRn)and complement dependent cytotoxicity via the Clq component of thecomplement cascade. The human IgG2 constant region lacks the ability toactivate complement by the classical pathway or to mediateantibody-dependent cellular cytotoxicity. The IgG4 constant region lacksthe ability to activate complement by the classical pathway and mediatesantibody-dependent cellular cytotoxicity only weakly. Antibodiesessentially lacking these effector functions may be termed ‘non-lytic’antibodies.

1.1.1 Human Antibodies

Human antibodies may be produced by a number of methods known to thoseof skill in the art. Human antibodies can be made by the hybridomamethod using human myeloma or mouse-human heteromyeloma cells lines seeKozbor J. Immunol 133, 3001, (1984) and Brodeur, Monoclonal AntibodyProduction Techniques and Applications, pp 51-63 (Marcel Dekker Inc,1987). Alternative methods include the use of phage libraries ortransgenic mice both of which utilize human V region repertories (seeWinter G, (1994), Annu. Rev. Immunol 12, 433-455, Green L L (1999), J.Immunol. methods 231, 11-23).

Several strains of transgenic mice are now available wherein their mouseimmunoglobulin loci has been replaced with human immunoglobulin genesegments (see Tomizuka K, (2000) PNAS 97, 722-727; Fishwild D. M (1996)Nature Biotechnol. 14, 845-851, Mendez M J, 1997, Nature Genetics, 15,146-156). Upon antigen challenge such mice are capable of producing arepertoire of human antibodies from which antibodies of interest can beselected.

Of particular note is the Trimera™ system (see Eren R et al, (1998)Immunology 93:154-161) where human lymphocytes are transplanted intoirradiated mice, the Selected Lymphocyte Antibody System (SLAM, seeBabcook et al, PNAS (1996) 93:7843-7848) where human (or other species)lymphocytes are effectively put through a massive pooled in vitroantibody generation procedure followed by deconvulated, limitingdilution and selection procedure and the Xenomouse II™ (Abgenix Inc). Analternative approach is available from Morphotek Inc using theMorphodoma™ technology.

Phage display technology can be used to produce human antibodies (andfragments thereof), see McCafferty; Nature, 348, 552-553 (1990) andGriffiths A D et al (1994) EMBO 13:3245-3260. According to thistechnique antibody V domain genes are cloned in frame into either amajor or minor coat of protein gene of a filamentous bacteriophage suchas M13 or fd and displayed (usually with the aid of a helper phage) asfunctional antibody fragments on the surface of the phage particle.Selections based on the functional properties of the antibody result inselection of the gene encoding the antibody exhibiting those properties.The phage display technique can be used to select antigen specificantibodies from libraries made from human B cells taken from individualsafflicted with a disease or disorder described above or alternativelyfrom unimmunized human donors (see Marks; J. Mol. Bio. 222,581-597,1991). Where an intact human antibody is desired comprising a Fc domainit is necessary to reclone the phage displayed derived fragment into amammalian expression vectors comprising the desired constant regions andestablishing stable expressing cell lines.

The technique of affinity maturation (Marks; Bio/technol 10, 779-783(1992)) may be used to improve binding affinity wherein the affinity ofthe primary human antibody is improved by sequentially replacing the Hand L chain V regions with naturally occurring variants and selecting onthe basis of improved binding affinities. Variants of this techniquesuch as “epitope imprinting” are now also available see WO 93/06213. Seealso Waterhouse; Nucl. Acids Res 21, 2265-2266 (1993).

1.2 Chimaeric and Humanised Antibodies

The use of intact non-human antibodies in the treatment of humandiseases or disorders carries with it the now well established problemsof potential immunogenicity especially upon repeated administration ofthe antibody that is the immune system of the patient may recognise thenon-human intact antibody as non-self and mount a neutralising response.In addition to developing fully human antibodies (see above) varioustechniques have been developed over the years to overcome these problemsand generally involve reducing the composition of non-human amino acidsequences in the intact therapeutic antibody whilst retaining therelative ease in obtaining non-human antibodies from an immunised animale.g. mouse, rat or rabbit. Broadly two approaches have been used toachieve this. The first are chimaeric antibodies, which generallycomprise a non-human (e.g. rodent such as mouse) variable domain fusedto a human constant region. Because the antigen-binding site of anantibody is localised within the variable regions the chimaeric antibodyretains its binding affinity for the antigen but acquires the effectorfunctions of the human constant region and are therefore able to performeffector functions such as described supra. Chimaeric antibodies aretypically produced using recombinant DNA methods. DNA encoding theantibodies (e.g. cDNA) is isolated and sequenced using conventionalprocedures (e.g. by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the H and L chain variableregions of the antibody of the invention, e.g. DNA of SEQ. I.D. NO 18and 20 described supra). Hybridoma cells serve as a typical source ofsuch DNA. Once isolated, the DNA is placed into expression vectors whichare then transfected into host cells such as E. Coli, COS cells, CHOcells, PerC6 cells or myeloma cells that do not otherwise produceimmunoglobulin protein to obtain synthesis of the antibody.

The DNA may be modified by substituting the coding sequence for human Land H chains for the corresponding non-human (e.g. murine) H and Lconstant regions see e.g. Morrison; PNAS 81, 6851 (1984). Thus anotherembodiment of the invention there is provided a chimaeric antibodycomprising a V_(H) domain having the sequence: SEQ ID No:17 and a V_(L)domain having the sequence: SEQ ID No: 19 fused to a human constantregion (which maybe of a IgG isotype e.g. IgG1).

The second approach involves the generation of humanised antibodieswherein the non-human content of the antibody is reduced by humanizingthe variable regions.

Two techniques for humanisation have gained popularity. The first ishumanisation by CDR grafting. CDRs build loops close to the antibody'sN-terminus where they form a surface mounted in a scaffold provided bythe framework regions. Antigen-binding specificity of the antibody ismainly defined by the topography and by the chemical characteristics ofits CDR surface. These features are in turn determined by theconformation of the individual CDRs, by the relative disposition of theCDRs, and by the nature and disposition of the side chains of theresidues comprising the CDRs. A large decrease in immunogenicity can beachieved by grafting only the CDRs of a non-human (e.g. murine)antibodies (“donor” antibodies) onto a suitable human framework(“acceptor framework”) and constant regions (see Jones et al (1986)Nature 321, 522-525 and Verhoeyen M et al (1988) Science 239,1534-1536). However, CDR grafting per se may not result in the completeretention of antigen-binding properties and it is frequently found thatsome framework residues of the donor antibody need to be preserved(sometimes referred to as “backmutations”) in the humanised molecule ifsignificant antigen-binding affinity is to be recovered (see Queen C etal (1989) PNAS 86, 10,029-10,033, Co, M et al (1991) Nature 351,501-502). In this case, human V regions showing the greatest sequencehomology (typically 60% or greater) to the non-human donor antibodymaybe chosen from a database in order to provide the human framework(FR). The selection of human FRs can be made either from human consensusor individual human antibodies. Where necessary key residues from thedonor antibody are substituted into the human acceptor framework topreserve CDR conformations. Computer modelling of the antibody maybeused to help identify such structurally important residues, seeWO99/48523.

Alternatively, humanisation maybe achieved by a process of “veneering”.A statistical analysis of unique human and murine immunoglobulin heavyand light chain variable regions revealed that the precise patterns ofexposed residues are different in human and murine antibodies, and mostindividual surface positions have a strong preference for a small numberof different residues (see Padlan E. A. et al; (1991) Mol. Immunol. 28,489-498 and Pedersen J. T. et al (1994) J. Mol. Biol. 235; 959-973).Therefore it is possible to reduce the immunogenicity of a non-human Fvby replacing exposed residues in its framework regions that differ fromthose usually found in human antibodies. Because protein antigenicitycan be correlated with surface accessibility, replacement of the surfaceresidues may be sufficient to render the mouse variable region“invisible” to the human immune system (see also Mark G. E. et al (1994)in Handbook of Experimental Pharmacology vol. 113: The pharmacology ofmonoclonal Antibodies, Springer-Verlag, pp 105-134). This procedure ofhumanisation is referred to as “veneering” because only the surface ofthe antibody is altered, the supporting residues remain undisturbed.Further alternative approaches include that set out in WO04/006955 andthe procedure of Humaneering™ (Kalobios) which makes use of bacterialexpression systems and produces antibodies that are close to humangermline in sequence (Alfenito-M Advancing Protein Therapeutics January2007, San Diego, Calif.).

It will be apparent to those skilled in the art that the term “derived”is intended to define not only the source in the sense of it being thephysical origin for the material but also to define material which isstructurally identical to the material but which does not originate fromthe reference source. Thus “residues found in the donor antibody” neednot necessarily have been purified from the donor antibody.

It is well recognised in the art that certain amino acid substitutionsare regarded as being “conservative”. Amino acids are divided intogroups based on common side-chain properties and substitutions withingroups that maintain all or substantially all of the binding affinity ofthe therapeutic antibody of the invention are regarded as conservativesubstitutions, see the following Table 1:

TABLE 1 Side chain Members Hydrophobic met, ala, val, leu, ile neutralhydrophilic cys, ser, thr Acidic asp, glu Basic asn, gln, his, lys, argresidues that influence chain gly, pro orientation aromatic trp, tyr,phe

1.3 Bispecific Antibodies

A bispecific antibody is an antibody derivative having bindingspecificities for at least two different epitopes and also forms part ofthe invention. Methods of making such antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin H chain-L chain pairs,where the two H chains have different binding specificities seeMillstein et al, Nature 305 537-539 (1983), WO93/08829 and Traunecker etal EMBO, 10, 1991, 3655-3659. Because of the random assortment of H andL chains, a potential mixture of ten different antibody structures areproduced of which only one has the desired binding specificity. Analternative approach involves fusing the variable domains with thedesired binding specificities to heavy chain constant region comprisingat least part of the hinge region, CH2 and CH3 regions. It is preferredto have the CH1 region containing the site necessary for light chainbinding present in at least one of the fusions. DNA encoding thesefusions, and if desired the L chain are inserted into separateexpression vectors and are then cotransfected into a suitable hostorganism. It is possible though to insert the coding sequences for twoor all three chains into one expression vector. In one preferredapproach, the bispecific antibody is composed of a H chain with a firstbinding specificity in one arm and a H-L chain pair, providing a secondbinding specificity in the other arm, see WO94/04690. See also Suresh etal Methods in Enzymology 121, 210, 1986.

Delivery of therapeutic proteins to the brain has been hampered by thepresence of the blood brain barrier (BBB). Where it is desired todeliver an antibody of the invention or antibody fragment of theinvention across the BBB various strategies have been proposed toenhance such delivery where needed.

In order to obtain required nutrients and factors from the blood, theBBB posseses some specific receptors, which transport compounds from thecirculating blood to the brain. Studies have indicated that somecompounds like insulin (see Duffy K R et al (1989) Brain Res.420:32-38), transferin (see Fishman J B et al (1987) J. Neurosci18:299-304) and insulin like growth factors 1 and 2 (see Pardridge W M(1986) Endocrine Rev. 7:314-330 and Duffy K R et al (1986) Metabolism37:136-140) traverse the BBB via receptor-mediated transcytosis.Receptors for these molecules thus provide a potential means fortherapeutic antibodies of the invention to access the brain usingso—called “vectored” antibodies (see Pardridge W M (1999) Advanced DrugDelivery Review 36:299-321). For example, an antibody to transferrinreceptor has been shown to be dynamically transported into the brainparenchyma (see Friden P M et al (1991) PNAS 88:4771-4775 and Friden P Met al (1993) Science 259:373-377). Thus one potential approach is toproduce a bispecific antibody or bispecific fragment such as describedsupra wherein a first specificity is towards and a second specificitytowards a transport receptor located at the BBB e.g. a secondspecificity towards the transferrin transport receptor.

1.4 Antibody Fragments

In certain embodiments of the invention there is provided therapeuticantibody which is an antigen binding fragment. Such fragments may befunctional antigen binding fragments of intact and/or humanised and/orchimaeric antibodies such as Fab, Fd, Fab′, F(ab′)₂, Fv, ScFv fragmentsof the antibodies described supra. Fragments lacking the constant regionlack the ability to activate complement by the classical pathway or tomediate antibody-dependent cellular cytotoxicity. Traditionally suchfragments are produced by the proteolytic digestion of intact antibodiesby e.g. papain digestion (see for example, WO 94/29348) but may beproduced directly from recombinantly transformed host cells. For theproduction of ScFv, see Bird et al; (1988) Science, 242, 423-426. Inaddition, antibody fragments may be produced using a variety ofengineering techniques as described below.

Fv fragments appear to have lower interaction energy of their two chainsthan Fab fragments. To stablise the association of the V_(H) and V_(L)domains, they have been linked with peptides (Bird et al, (1988) Science242, 423-426, Huston et al, PNAS, 85, 5879-5883), disulphide bridges(Glockshuber et al, (1990) Biochemistry, 29, 1362-1367) and “knob inhole” mutations (Zhu et al (1997), Protein Sci., 6, 781-788). ScFvfragments can be produced by methods well known to those skilled in theart see Whitlow et al (1991) Methods companion Methods Enzymol, 2,97-105 and Huston et al (1993) Int. Rev. Immunol 10, 195-217. ScFv maybe produced in bacterial cells such as E. Coli but are more typicallyproduced in eukaryotic cells.

One disadvantage of ScFv is the monovalency of the product, whichprecludes an increased avidity due to polyvalent binding, and theirshort half-life. Attempts to overcome these problems include bivalent(ScFv′)₂ produced from ScFV containing an additional C terminal cysteineby chemical coupling (Adams et al (1993) Can. Res 53, 4026-4034 andMcCartney et al (1995) Protein Eng. 8, 301-314) or by spontaneoussite-specific dimerization of ScFv containing an unpaired C terminalcysteine residue (see Kipriyanov et al (1995) Cell. Biophys 26,187-204). Alternatively, ScFv can be forced to form multimers byshortening the peptide linker to between 3 to 12 residues to form“diabodies”, see Holliger et al PNAS (1993), 90, 6444-6448. Reducing thelinker still further can result in ScFV trimers (“triabodies”, see Korttet al (1997) Protein Eng, 10, 423-433) and tetramers (“tetrabodies”, seeLe Gall et al (1999) FEBS Lett, 453, 164-168). Construction of bivalentScFV molecules can also be achieved by genetic fusion with proteindimerizing motifs to form “miniantibodies” (see Pack et al (1992)Biochemistry 31, 1579-1584) and “minibodies” (see Hu et al (1996),Cancer Res. 56, 3055-3061). ScFv-Sc-Fv tandems ((ScFV)₂) may also beproduced by linking two ScFv units by a third peptide linker, see Kuruczet al (1995) J. Immol. 154, 4576-4582. Bispecific diabodies can beproduced through the noncovalent association of two single chain fusionproducts consisting of V_(H) domain from one antibody connected by ashort linker to the V_(L) domain of another antibody, see Kipriyanov etal (1998), Int. J. Can 77, 763-772. The stability of such bispecificdiabodies can be enhanced by the introduction of disulphide bridges or“knob in hole” mutations as described supra or by the formation ofsingle chain diabodies (ScDb) wherein two hybrid ScFv fragments areconnected through a peptide linker see Kontermann et al (1999) J.Immunol. Methods 226 179-188. Tetravalent bispecific molecules areavailable by e.g. fusing a ScFv fragment to the CH3 domain of an IgGmolecule or to a Fab fragment through the hinge region see Coloma et al(1997) Nature Biotechnol. 15, 159-163. Alternatively, tetravalentbispecific molecules have been created by the fusion of bispecificsingle chain diabodies (see Alt et al, (1999) FEBS Lett 454, 90-94.Smaller tetravalent bispecific molecules can also be formed by thedimerization of either ScFv-ScFv tandems with a linker containing ahelix-loop-helix motif (DiBi miniantibodies, see Muller et al (1998)FEBS Lett 432, 45-49) or a single chain molecule comprising fourantibody variable domains (V_(H) and V_(L)) in an orientation preventingintramolecular pairing (tandem diabody, see Kipriyanov et al, (1999) J.Mol. Biol. 293, 41-56). Bispecific F(ab′)2 fragments can be created bychemical coupling of Fab′ fragments or by heterodimerization throughleucine zippers (see Shalaby et al, (1992) J. Exp. Med. 175, 217-225 andKostelny et al (1992), J. Immunol. 148, 1547-1553). Also available areisolated V_(H) and V_(L) domains, see U.S. Pat. No. 6,248,516; U.S. Pat.No. 6,291,158; U.S. Pat. No. 6,172,197.

1.5 Heteroconjugate Antibodies

Heteroconjugate antibodies are derivatives which also form an embodimentof the present invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies formed using any convenient cross-linkingmethods. See U.S. Pat. No. 4,676,980.

1.6 Other Modifications.

The interaction between the Fc region of an antibody and various Fcreceptors (FcγR) is believed to mediate the effector functions of theantibody which include antibody-dependent cellular cytotoxicity (ADCC),fixation of complement, phagocytosis and half-life/clearance of theantibody. Various modifications to the Fc region of antibodies of theinvention may be carried out depending on the desired effector property.In particular, human constant regions which essentially lack thefunctions of a) activation of complement by the classical pathway; andb) mediating antibody-dependent cellular cytotoxicity include the IgG4constant region, the IgG2 constant region and IgG1 constant regionscontaining specific mutations as for example mutations at positions 234,235, 236, 237, 297, 318, 320 and/or 322 disclosed in EP0307434(WO8807089), EP 0629 240 (WO9317105) and WO 2004/014953. Mutations atresidues 235 or 237 within the CH2 domain of the heavy chain constantregion (Kabat numbering; EU Index system) have separately been describedto reduce binding to FcγRI, FcγRII and FcγRIII binding and thereforereduce antibody-dependent cellular cytotoxicity (ADCC) (Duncan et al.Nature 1988, 332; 563-564; Lund et al. J. Immunol. 1991, 147; 2657-2662;Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. Immunol.1992, 51; 1-84; Morgan et al., Immunology 1995, 86; 319-324; Hezareh etal., J. Virol. 2001, 75 (24); 12161-12168). Further, some reports havealso described involvement of some of these residues in recruiting ormediating complement dependent cytotoxicity (CDC) (Morgan et al., 1995;Xu et al., Cell. Immunol. 2000; 200:16-26; Hezareh et al., J. Virol.2001, 75 (24); 12161-12168). Residues 235 and 237 have therefore bothbeen mutated to alanine residues (Brett et al. Immunology 1997, 91;346-353; Bartholomew et al. Immunology 1995, 85; 41-48; and WO9958679)to reduce both complement mediated and FcγR-mediated effects. Antibodiescomprising these constant regions may be termed ‘non-lytic’ antibodies.

One may incorporate a salvage receptor binding epitope into the antibodyto increase serum half life see U.S. Pat. No. 5,739,277.

There are five currently recognised human Fcγ receptors, FcγR (I),FcγRIIa, FcγRIIb, FcγRIIIa and neonatal FcRn. Shields et al, (2001) J.Biol. Chem. 276, 6591-6604 demonstrated that a common set of IgG1residues is involved in binding all FcγR5, while FcγRII and FcγRIIIutilize distinct sites outside of this common set. One group of IgG1residues reduced binding to all FcγR5 when altered to alanine: Pro-238,Asp-265, Asp-270, Asn-297 and Pro-239. All are in the IgG CH2 domain andclustered near the hinge joining CH1 and CH2. While FcγRI utilizes onlythe common set of IgG1 residues for binding, FcγRII and FcγRIII interactwith distinct residues in addition to the common set. Alteration of someresidues reduced binding only to FcγRII (e.g. Arg-292) or FcγRIII (e.g.Glu-293). Some variants showed improved binding to FcγRII or FcγRIII butdid not affect binding to the other receptor (e.g. Ser-267Ala improvedbinding to FcγRII but binding to FcγRIII was unaffected). Other variantsexhibited improved binding to FcγRII or FcγRIII with reduction inbinding to the other receptor (e.g. Ser-298Ala improved binding toFcγRIII and reduced binding to FcγRII). For FcγRIIIa, the best bindingIgG1 variants had combined alanine substitutions at Ser-298, Glu-333 andLys-334. The neonatal FcRn receptor is believed to be involved inprotecting IgG molecules from degradation and thus enhancing serum halflife and the transcytosis across tissues (see Junghans R. P (1997)Immunol. Res 16. 29-57 and Ghetie et al (2000) Annu. Rev. Immunol. 18,739-766). Human IgG1 residues determined to interact directly with humanFcRn includes 11e253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435.

The therapeutic antibody of the invention may incorporate any of theabove constant region modifications.

In a particular embodiment, the therapeutic antibody essentially lacksthe functions of a) activation of complement by the classical pathway;and b) mediating antibody-dependent cellular cytotoxicity. In a moreparticular embodiment the present invention provides therapeuticantibodies of the invention having any one (or more) of the residuechanges detailed above to modify half-life/clearance and/or effectorfunctions such as ADCC and/or complement dependent cytotoxicity and/orcomplement lysis.

In a further aspect of the present invention the therapeutic antibodyhas a constant region of isotype human IgG1 with alanine (or otherdisrupting) substitutions at positions 235 (e.g. L235A) and 237 (e.g.G237A) (numbering according to the EU scheme outlined in Kabat.

Other derivatives of the invention include glycosylation variants of theantibodies of the invention. Glycosylation of antibodies at conservedpositions in their constant regions is known to have a profound effecton antibody function, particularly effector functioning such as thosedescribed above, see for example, Boyd et al (1996), Mol. Immunol. 32,1311-1318. Glycosylation variants of the therapeutic antibodies of thepresent invention wherein one or more carbohydrate moiety is added,substituted, deleted or modified are contemplated. Introduction of anasparagine-X-serine or asparagine-X-threonine motif creates a potentialsite for enzymatic attachment of carbonhydrate moieties and maytherefore be used to manipulate the glycosylation of an antibody. InRaju et al (2001) Biochemistry 40, 8868-8876 the terminal sialyation ofa TNFR-IgG immunoadhesin was increased through a process ofregalactosylation and/or resialylation usingbeta-1,4-galactosyltransferace and/or alpha, 2,3 sialyltransferase.Increasing the terminal sialylation is believed to increase thehalf-life of the immunoglobulin. Antibodies, in common with mostglycoproteins, are typically produced in nature as a mixture ofglycoforms. This mixture is particularly apparent when antibodies areproduced in eukaryotic, particularly mammalian cells. A variety ofmethods have been developed to manufacture defined glycoforms, see Zhanget al Science (2004), 303, 371, Sears et al, Science, (2001) 291, 2344,Wacker et al (2002) Science, 298 1790, Davis et al (2002) Chem. Rev.102, 579, Hang et al (2001) Acc. Chem. Res 34, 727. Thus the inventionconcerns a plurality of therapeutic antibodies (which maybe of the IgGisotype, e.g. IgG1) as described herein comprising a defined number(e.g. 7 or less, for example 5 or less such as two or a single)glycoform(s) of said antibodies.

Derivatives according to the invention also include therapeuticantibodies of the invention coupled to a non-proteinaeous polymer suchas polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.Conjugation of proteins to PEG is an established technique forincreasing half-life of proteins, as well as reducing antigenicity andimmunogenicity of proteins. The use of PEGylation with differentmolecular weights and styles (linear or branched) has been investigatedwith intact antibodies as well as Fab′ fragments, see Koumenis I. L. etal (2000) Int. J. Pharmaceut. 198:83-95. A particular embodimentcomprises an antigen-binding fragment of the invention without theeffector functions of a) activation of complement by the classicalpathway; and b) mediating antibody-dependent cellular cytotoxicity;(such as a Fab fragment or a scFv) coupled to PEG.

2. Production Methods

Antibodies of the present invention may be produced in transgenicorganisms such as goats (see Pollock et al (1999), J. Immunol. Methods231:147-157), chickens (see Morrow K J J (2000) Genet. Eng. News20:1-55), mice (see Pollock et al ibid) or plants (see Doran P M, (2000)Curr. Opinion Biotechnol. 11, 199-204, Ma J K-C (1998), Nat. Med. 4;601-606, Baez J et al, BioPharm (2000) 13: 50-54, Stoger E et al; (2000)Plant Mol. Biol. 42:583-590). Antibodies may also be produced bychemical synthesis. However, antibodies of the invention are typicallyproduced using recombinant cell culturing technology well known to thoseskilled in the art. A polynucleotide encoding the antibody is isolatedand inserted into a replicable vector such as a plasmid for furtherpropagation or expression in a host cell. One useful expression systemis a glutamate synthetase system (such as sold by Lonza Biologics),particularly where the host cell is CHO or NSO (see below).Polynucleotide encoding the antibody is readily isolated and sequencedusing conventional procedures (e.g. oligonucleotide probes). Vectorsthat may be used include plasmid, virus, phage, transposons,minichromsomes of which plasmids are a typical embodiment. Generallysuch vectors further include a signal sequence, origin of replication,one or more marker genes, an enhancer element, a promoter andtranscription termination sequences operably linked to the light and/orheavy chain polynucleotide so as to facilitate expression.Polynucleotide encoding the light and heavy chains may be inserted intoseparate vectors and introduced (e.g. by transformation, transfection,electroporation or transduction) into the same host cell concurrently orsequentially or, if desired both the heavy chain and light chain can beinserted into the same vector prior to such introduction.

It will be immediately apparent to those skilled in the art that due tothe redundancy of the genetic code, alternative polynucleotides to thosedisclosed herein are also available that will encode the polypeptides ofthe invention.

2.1 Signal Sequences

Antibodies of the present invention maybe produced as a fusion proteinwith a heterologous signal sequence having a specific cleavage site atthe N terminus of the mature protein. The signal sequence should berecognised and processed by the host cell. For prokaryotic host cells,the signal sequence may be an alkaline phosphatase, penicillinase, orheat stable enterotoxin II leaders. For yeast secretion the signalsequences may be a yeast invertase leader, a factor leader or acidphosphatase leaders see e.g. WO90/13646. In mammalian cell systems,viral secretory leaders such as herpes simplex gD signal and nativeimmunoglobulin signal sequences (such as human Ig heavy chain) areavailable. Typically the signal sequence is ligated in reading frame topolynucleotide encoding the antibody of the invention.

2.2 Origin of Replication Origin of replications are well known in theart with pBR322 suitable for most gram-negative bacteria, 2μ, plasmidfor most yeast and various viral origins such as SV40, polyoma,adenovirus, VSV or BPV for most mammalian cells. Generally the SV40origin of replication component is not needed for integrated mammalianexpression vectors. However the SV40 on may be included since itcontains the early promoter.

2.3 Selection Marker

Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins e.g. ampicillin, neomycin, methotrexate ortetracycline or (b) complement auxotrophic deficiencies or supplynutrients not available in the complex media or (c) combinations ofboth. The selection scheme may involve arresting growth of the hostcells that contain no vector or vectors. Cells, which have beensuccessfully transformed with the genes encoding the therapeuticantibody of the present invention, survive due to e.g. drug resistanceconferred by the co-delivered selection marker. One example is theDHFR-selection system wherein transformants are generated in DHFRnegative host strains (eg see Page and Sydenham 1991 Biotechnology 9:64-68). In this system the DHFR gene is co-delivered with antibodypolynucleotide sequences of the invention and DHFR positive cells thenselected by nucleoside withdrawal. If required, the DHFR inhibitormethotrexate is also employed to select for transformants with DHFR geneamplification. By operably linking DHFR gene to the antibody codingsequences of the invention or functional derivatives thereof, DHFR geneamplification results in concomitant amplification of the desiredantibody sequences of interest. CHO cells are a particularly useful cellline for this DHFR/methotrexate selection and methods of amplifying andselecting host cells using the DHFR system are well established in theart see Kaufman R. J. et al J. Mol. Biol. (1982) 159, 601-621, forreview, see Werner R G, Noe W, Kopp K, Schluter M, “Appropriatemammalian expression systems for biopharmaceuticals”,Arzneimittel-Forschung. 48(8):870-80, 1998 August A further example isthe glutamate synthetase expression system (Bebbington et alBiotechnology 1992 Vol 10 p169). A suitable selection gene for use inyeast is the trp1 gene; see Stinchcomb et al Nature 282, 38, 1979.

2.4 Promoters

Suitable promoters for expressing antibodies of the invention areoperably linked to DNA/polynucleotide encoding the antibody. Promotersfor prokaryotic hosts include phoA promoter, Beta-lactamase and lactosepromoter systems, alkaline phosphatase, tryptophan and hybrid promoterssuch as Tac. Promoters suitable for expression in yeast cells include3-phosphoglycerate kinase or other glycolytic enzymes e.g. enolase,glyceralderhyde 3 phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose 6 phosphate isomerase,3-phosphoglycerate mutase and glucokinase. Inducible yeast promotersinclude alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,metallothionein and enzymes responsible for nitrogen metabolism ormaltose/galactose utilization. Promoters for expression in mammaliancell systems include RNA polymerase II promoters including viralpromoters such as polyoma, fowlpox and adenoviruses (e.g. adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus (inparticular the immediate early gene promoter), retrovirus, hepatitis Bvirus, actin, rous sarcoma virus (RSV) promoter and the early or lateSimian virus 40 and non-viral promoters such as EF-1alpha (Mizushima andNagata Nucleic Acids Res 1990 18(17):5322. The choice of promoter may bebased upon suitable compatibility with the host cell used forexpression.

2.5 Enhancer Element

Where appropriate, e.g. for expression in higher eukaroytics, additionalenhancer elements can included instead of or as well as those foundlocated in the promoters described above. Suitable mammalian enhancersequences include enhancer elements from globin, elastase, albumin,fetoprotein, metallothionine and insulin. Alternatively, one may use anenhancer element from a eukaroytic cell virus such as SV40 enhancer,cytomegalovirus early promoter enhancer, polyoma enhancer, baculoviralenhancer or murine IgG2a locus (see WO04/009823). Whilst such enhancersare typically located on the vector at a site upstream to the promoter,they can also be located elsewhere e.g. within the untranslated regionor downstream of the polydenalytion signal. The choice and positioningof enhancer may be based upon suitable compatibility with the host cellused for expression.

2.6 Polyadenylation/Termination

In eukaryotic systems, polyadenylation signals are operably linked topolynucleotide encoding the antibody of this invention. Such signals aretypically placed 3′ of the open reading frame. In mammalian systems,non-limiting example signals include those derived from growth hormones,elongation factor-1 alpha and viral (eg SV40) genes or retroviral longterminal repeats. In yeast systems non-limiting examples ofpolydenylation/termination signals include those derived from thephosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1 (ADH)genes. In prokaryotic system polyadenylation signals are typically notrequired and it is instead usual to employ shorter and more definedterminator sequences. The choice of polyadenylation/terminationsequences may be based upon suitable compatibility with the host cellused for expression.

2.7 Other Methods/Elements for Enhanced Yields

In addition to the above, other features that can be employed to enhanceyields include chromatin remodelling elements, introns and host-cellspecific codon modification. The codon useage of the antibody of thisinvention thereof can be modified to accommodate codon bias of the hostcell such to augment transcript and/or product yield (eg Hoekema A et alMol Cell Biol 1987 7(8):2914-24). The choice of codons may be based uponsuitable compatibility with the host cell used for expression.

2.8 Host Cells

Suitable host cells for cloning or expressing vectors encodingantibodies of the invention are prokaroytic, yeast or higher eukaryoticcells. Suitable prokaryotic cells include eubacteria e.g.enterobacteriaceae such as Escherichia e.g. E. Coli (for example ATCC31,446; 31,537; 27,325), Enterobacter, Erwinia, Klebsiella Proteus,Salmonella e.g. Salmonella typhimurium, Serratia e.g. Serratiamarcescans and Shigella as well as Bacilli such as B. subtilis and B.licheniformis (see DD 266 710), Pseudomonas such as P. aeruginosa andStreptomyces. Of the yeast host cells, Saccharomyces cerevisiae,schizosaccharomyces pombe, Kluyveromyces (e.g. ATCC 16,045; 12,424;24178; 56,500), yarrowia (EP402, 226), Pichia Pastoris (EP183, 070, seealso Peng et al J. Biotechnol. 108 (2004) 185-192), Candida, Trichodermareesia (EP244, 234), Penicillin, Tolypocladium and Aspergillus hostssuch as A. nidulans and A. niger are also contemplated.

Although Prokaryotic and yeast host cells are specifically contemplatedby the invention, typically however, host cells of the present inventionare vertebrate cells. Suitable vertebrate host cells include mammaliancells such as COS-1 (ATCC No. CRL 1650) COS-7 (ATCC CRL 1651), humanembryonic kidney line 293, PerC6 (Crucell), baby hamster kidney cells(BHK) (ATCC CRL. 1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCC NO. CRL1573), Chinese hamster ovary cells CHO (e.g. CHO-K1, ATCC NO: CCL 61,DHFR minus CHO cell line such as DG44 (Urlaub et al, Somat Cell MolGenet (1986) Vol 12 pp 555-566), particularly those CHO cell linesadapted for suspension culture, mouse sertoli cells, monkey kidneycells, African green monkey kidney cells (ATCC CRL-1587), HELA cells,canine kidney cells (ATCC CCL 34), human lung cells (ATCC CCL 75), HepG2 and myeloma or lymphoma cells e.g. NS0 (see U.S. Pat. No. 5,807,715),Sp2/0, Y0.

Thus in one embodiment of the invention there is provided a stablytransformed host cell comprising a vector encoding a heavy chain and/orlight chain of the therapeutic antibody as described herein. Typicallysuch host cells comprise a first vector encoding the light chain and asecond vector encoding said heavy chain.

Such host cells may also be further engineered or adapted to modifyquality, function and/or yield of the antibody of this invention.Non-limiting examples include expression of specific modifying (egglycosylation) enzymes and protein folding chaperones.

2.9 Cell Culturing Methods.

Host cells transformed with vectors encoding the therapeutic antibodiesof the invention may be cultured by any method known to those skilled inthe art. Host cells may be cultured in spinner flasks, shake flasks,roller bottles, wave reactors (eg System 1000 from wavebiotech.com) orhollow fibre systems but it is preferred for large scale production thatstirred tank reactors or bag reactors (eg Wave Biotech, Somerset, N.J.USA) are used particularly for suspension cultures. Typically thestirred tankers are adapted for aeration using e.g. spargers, baffles orlow shear impellers. For bubble columns and airlift reactors directaeration with air or oxygen bubbles maybe used. Where the host cells arecultured in a serum free culture media this can be supplemented with acell protective agent such as pluronic F-68 to help prevent cell damageas a result of the aeration process. Depending on the host cellcharacteristics, either microcarriers maybe used as growth substratesfor anchorage dependent cell lines or the cells maybe adapted tosuspension culture (which is typical). The culturing of host cells,particularly vertebrate host cells may utilise a variety of operationalmodes such as batch, fed-batch, repeated batch processing (see Drapeauet al (1994) cytotechnology 15: 103-109), extended batch process orperfusion culture. Although recombinantly transformed mammalian hostcells may be cultured in serum-containing media such media comprisingfetal calf serum (FCS), it is preferred that such host cells arecultured in serum—free media such as disclosed in Keen et al (1995)Cytotechnology 17:153-163, or commercially available media such asProCHO-CDM or UltraCHO™ (Cambrex NJ, USA), supplemented where necessarywith an energy source such as glucose and synthetic growth factors suchas recombinant insulin. The serum-free culturing of host cells mayrequire that those cells are adapted to grow in serum free conditions.One adaptation approach is to culture such host cells in serumcontaining media and repeatedly exchange 80% of the culture medium forthe serum-free media so that the host cells learn to adapt in serum freeconditions (see e.g. Scharfenberg K et al (1995) in Animal Celltechnology: Developments towards the 21st century (Beuvery E. C. et aleds), pp 619-623, Kluwer Academic publishers).

Antibodies of the invention secreted into the media may be recovered andpurified from the media using a variety of techniques to provide adegree of purification suitable for the intended use. For example theuse of therapeutic antibodies of the invention for the treatment ofhuman patients typically mandates at least 95% purity as determined byreducing SDS-PAGE, more typically 98% or 99% purity, when compared tothe culture media comprising the therapeutic antibodies. In the firstinstance, cell debris from the culture media is typically removed usingcentrifugation followed by a clarification step of the supernatant usinge.g. microfiltration, ultrafiltration and/or depth filtration.Alternatively, the antibody can be harvested by microfiltration,ultrafiltration or depth filtration without prior centrifugation. Avariety of other techniques such as dialysis and gel electrophoresis andchromatographic techniques such as hydroxyapatite (HA), affinitychromatography (optionally involving an affinity tagging system such aspolyhistidine) and/or hydrophobic interaction chromatography (HIC, seeU.S. Pat. No. 5,429,746) are available. In one embodiment, theantibodies of the invention, following various clarification steps, arecaptured using Protein A or G affinity chromatography followed byfurther chromatography steps such as ion exchange and/or HAchromatography, anion or cation exchange, size exclusion chromatographyand ammonium sulphate precipitation. Typically, various virus removalsteps are also employed (e.g. nanofiltration using e.g. a DV-20 filter).Following these various steps, a purified (typically monoclonal)preparation comprising at least 10 mg/ml or greater e.g. 100 mg/ml orgreater of the antibody of the invention is provided and therefore formsan embodiment of the invention. Concentration to 100 mg/ml or greatercan be generated by ultracentrifugation. Suitably such preparations aresubstantially free of aggregated forms of antibodies of the invention.

Bacterial systems are particularly suited for the expression of antibodyfragments. Such fragments are localised intracellularly or within theperiplasma. Insoluble periplasmic proteins can be extracted and refoldedto form active proteins according to methods known to those skilled inthe art, see Sanchez et al (1999) J. Biotechnol. 72, 13-20 and Cupit P Met al (1999) Lett Appl Microbiol, 29, 273-277.

3. Pharmaceutical Compositions

Purified preparations of antibodies of the invention (particularlymonoclonal preparations) as described supra, may be incorporated intopharmaceutical compositions for use in the treatment of human diseasesand disorders such as those outlined above. Typically such compositionsfurther comprise a pharmaceutically acceptable (i.e. inert) carrier asknown and called for by acceptable pharmaceutical practice, see e.g.Remingtons Pharmaceutical Sciences, 16th ed, (1980), Mack Publishing Co.Examples of such carriers include sterilised carrier such as saline,Ringers solution or dextrose solution, buffered with suitable bufferssuch as sodium acetate trihydrate to a pharmaceutically acceptable pH,such as a pH within a range of 5 to 8. Pharmaceutical compositons forinjection (e.g. by intravenous, intraperitoneal, intradermal,subcutaneous, intramuscular or intraportal) or continuous infusion aresuitably free of visible particulate matter and may comprise from 1 mgto 10 g of therapeutic antibody, typically 5 mg to 1 g, morespecifically 5 mg to 25 mg or 50 mg of antibody. Methods for thepreparation of such pharmaceutical compositions are well known to thoseskilled in the art. In one embodiment, pharmaceutical compositionscomprise from 1 mg to 10 g of therapeutic antibodies of the invention inunit dosage form, optionally together with instructions for use.Pharmaceutical compositions of the invention may be lyophilised (freezedried) for reconstitution prior to administration according to methodswell known or apparent to those skilled in the art. Where embodiments ofthe invention comprise antibodies of the invention with an IgG1 isotype,a chelator of metal ions including copper, such as citrate (e.g. sodiumcitrate) or EDTA or histidine, may be added to the pharmaceuticalcomposition to reduce the degree of metal-mediated degradation ofantibodies of this isotype, see EP0612251. Pharmaceutical compositionsmay also comprise a solubiliser such as arginine base, adetergent/anti-aggregation agent such as polysorbate 80, and an inertgas such as nitrogen to replace vial headspace oxygen.

Effective doses and treatment regimes for administering the antibody ofthe invention are generally determined empirically and are dependent onfactors such as the age, weight and health status of the patient anddisease or disorder to be treated. Such factors are within the purviewof the attending physican. Guidance in selecting appropriate doses maybe found in e.g. Smith et al (1977) Antibodies in human diagnosis andtherapy, Raven Press, New York but will in general be 1 mg to 10 g. Inone embodiment, the dosing regime for treating a human patient is 1 mgto 1 g of therapeutic antibody of the invention administeredsubcutaneously once per week or every two weeks, or by intravenousinfusion every 1 or 2 months. Such a dosage corresponds to 0.014-140mg/kg, such as 0.014-14 mg/kg. Compositions of the present invention mayalso be used prophylatically.

4. Clinical Uses.

It will be appreciated that diseases characterised by elevated β-amyloidlevels or β-amyloid deposits include Alzheimer's disease, mild cognitiveimpairment, Down's syndrome, hereditary cerebral haemorrhage withβ-amyloidosis of the Dutch type, cerebral β-amyloid angiopathy andvarious types of degenerative dementias, such as those associated withParkinson's disease, progressive supranuclear palsy, cortical basaldegeneration and diffuse Lewis body type of Alzheimer's disease.

Most preferably, the disease characterised by elevated β-amyloid levelsor β-amyloid deposits is Alzheimer's disease.

Although the present invention has been described principally inrelation to the treatment of human diseases or disorders, the presentinvention may also have applications in the treatment of similardiseases or disorders in non-human mammals.

EXAMPLES Methods

-   Biacore™/Biacore 3000 a device that allows measurement of real time    kinetics of molecular interactions using SPR-   SPR (surface plasmon resonance)—physical phenomenon employed by    Biacore instruments for measurement of mass changes on sensor chip-   CM5 Biacore™ sensor chip with general purpose surface coated with a    carboxymethylated dextran matrix ELISA enzyme linked immunosorbent    assay-   SRU SRU BIND™ Biosensor technology allowing to monitor label-free    biochemical interactions-   Integra CL1000 Mini-bioreactors sold by IBS Integra Biosciences-   FMAT fluorometric microvolume assay technology (Applied Biosystems)-   ABi8200 Applied Biosystems 8200 fluorescence macro confocal cellular    detection system for FMAT-   FPLC Fast protein liquid chromatography-   ProSepA HiTrap Protein A columns for FPLC sold by GE Healthcare

Materials

-   DMSO dimethylsulphoxide-   HEPES N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)-   EDTA ethylenediaminetetraacetic acid-   Tris HCl—tris-(hydroxymethyl)aminomethane hydrochloride-   NaCl—sodium chloride-   Tween-20—polyoxyethylenesorbitan monolaurate-   BSA—bovine serum albumin-   PBS—phosphate buffered saline-   PFA—paraformaldehyde-   IMS—industrial methylated spirit-   DAB—3,3′ diaminobenzidine-   DMEM dulbecco's modified eagle's medium-   FCS fetal calf serum-   Opti-MEM modified eagle's medium based medium by Invitrogen/Gibco-   Lipofectamine cationic lipid based cell transfection agent sold by    Invitrogen/Gibco-   Transfast liposomal transfection agent sold by Promega-   Versene metal ion chelating agent (ethylenediaminetetraacetic acid)-   Glutamax stable form of glutamine added to culture medium (dipeptide    L-Ananyl-L-Glutamine supplement)-   Histoclear tissue clearing agent-   HBS-EP buffer General purpose Biacore™ buffer containing 0.01M HEPES    pH7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20

Generation of Mouse Monoclonal Antibody 2E7

Mouse monoclonal antibody 2E7 was generated from a conventionalimmunisation of mice. Mice were immunised with soluble or aggregatedβ-amyloid 1-40 and 1-42 formulated in Freund's adjuvant. Following finalboost without adjuvant, splenocytes were fused with myeloma cells. Fusedcells were grown in 96-well plates from which hybridoma supernatantswere screened for potential leads. Selected antibody 2E7, which wasobtained from the immunisation with soluble β-amyloid 1-40, was ofmurine IgG2a isotype and had beta-amyloid binding activity in the effluxassay described below and an affinity of 36.1 μM for beta-amyloid 1-40when measured by Biacore™, Method A(i) (Table 10A).

Epitope Mapping of 2E7

In order to finely map the binding of antibody 2E7 to the β-amyloidpeptide, a peptide set (A) was utilised. Peptide set (A) consisted of aset of 31 12-mer overlapping peptides covering the complete sequence ofthe β-amyloid 1-42 peptide. Each sequential peptide was initiated at thesequential amino acid within the β-amyloid peptide, thus shifting thesequence covered between sequential peptides by a single amino acid. Allpeptides in set (A) contained a 3 amino acid C-terminal linker(glycine-serine-glycine) and a terminal biotinylated lysine residue. Inaddition, all peptides except peptide Aβ1 DAEFRHDSGYEVGSGK-biotin (SEQID No:15) were N-terminally acetylated. A second set of peptides (set(B)) consisted of sequential one amino acid C-terminal deletions from apeptide containing amino acids 1 to 10 of the β-amyloid sequence. Allpeptides in set (B) contained a 3 amino acid C-terminal linker(glycine-serine-glycine) and a terminal biotinylated lysine residue, butwith additional glycine and serine residues to replace for deletedβ-amyloid amino acids (Table 2). Thus all peptides in set (B) are of thesame length.

TABLE 2  Sequences of biotinylated peptides(set (B) that contained truncated  N-terminal fragments of β-amyloidDAEFRHDSGYGSGGSK-biotin (SEQ ID No: 7) DAEFRHDSG--GSGSGSK-biotin(SEQ ID No: 8) DAEFRHDS--GSGGSGGK-biotin (SEQ ID No: 9)DAEFRHD--GSGGSGGSK-biotin (SEQ ID No: 10) DAEFRH--GSGGSGGSGK-biotin(SEQ ID No: 11) DAEFR--GSGGSGGSGSK-biotin (SEQ ID No: 12)DAEF--GSGGSGGSGGSK-biotin (SEQ ID No: 13) DAE--GSGGSGGSGGSGK-biotin(SEQ ID No: 14)

Monitoring the Binding of 2E7 to β-Amyloid Derived Peptides UsingOptical Biosensors

96-well SRU Bind™ streptavidin-coated plates (SRU Biosystems) werewashed with PBS containing 1% DMSO to remove glycerol and preservative.A volume of 50 ul/well was left to equilibrate to room temperature toprovide a constant base line. Biotinylated peptides were diluted toapprox. 0.3 ug/ml in PBS containing 1% DMSO and 50 ul of each added towells and incubated for approximately 1 h. Replicate wells were preparedusing different sectors of the plate and at least one no-peptide controlwell was used in each sector to reference subtract the data. Afterpeptide capture the plate was washed with PBS containing 1% DMSO,leaving 50 ul of fresh buffer per well to provide a new base line on thereader. No decay of peptide from the surface was seen. The buffer wasthen replaced with 40 ul/well buffer containing test antibody at 20-64nM. for 2 hours. It was found that antibody 2E7 only bound to thepeptide encompassing amino acids 1-12 of the β-amyloid peptide inpeptide set (A) (peptide Aβ1, SEQ ID No:15) This result implies that theaspartic acid at residue 1 is critical for binding to this peptide.

In order to further characterise the binding site of antibody 2E7,peptide set (B) was utilised. Using SRU BIND™ biosensor methodologyantibody 2E7 showed negligible binding to the peptides encompassingamino acids 1-3 and 1-4 of the β-amyloid peptide (SEQ ID No:14 and 13).Binding to a peptide encompassing amino acids 1-7 of the β-amyloidpeptide (SEQ ID No:10) was comparable to the peptide encompassing aminoacids 1-12 of the β-amyloid peptide (from peptide set (A)). Binding topeptides encompassing amino acids 1-5 or 1-6 of the β-amyloid peptide(SEQ ID No:12 or 11) was observed, but was weaker (as measured bystability after an additional washing step) than the binding to thepeptide encompassing amino acids 1-7 of the β-amyloid peptide (SEQ IDNo:10).

Thus it has been shown that only residues 1-7 of the β-amyloid peptideare required for full binding as measured using this methodology.

Surface Plasmon Resonance assay

In addition to the experiments described above, the Biacore™ 3000optical biosensor was used to monitor the binding of 2E7 antibody toselected β-amyloid sequence derived peptides. Binding was measured byinjecting test antibodies at up to 64 nM for 5 minute over peptidescaptured on separate streptavidin chip surfaces (130-230 RU (resonanceunits)). A running buffer (HBS-EP) containing 0.01M HEPES pH7.4, 0.15MNaCl, 3 mM EDTA and 0.005% Surfactant P20™ at 25° C. was used at a flowrate of 20 ul/min. All runs were double referenced against a blankstreptavidin surface and blank injections. Analysis was carried outusing the Biacore analysis software BIAevaluation™ version 4.1. Resultsfrom selected peptides in set (A) further confirmed the SRU BIND™derived data indicating that 2E7 bound only to the peptide encompassingamino acids 1-12 (SEQ ID No:15) of the β-amyloid peptide with anapparent equilibrium constant KD of approximately 50 μM. Under the sameconditions, 2E7 did not bind to the peptide encompassing amino acids2-13 of the β-amyloid peptide.

Peptide Aβ2-13 (SEQ ID No: 44) AEFRHDSGYEVHGSGK-biotin

The experimental method and conditions used allowed the detection ofhigh but also lower affinity molecules—in the same experimental setup,by contrast to 2E7, another antibody recognising an N-terminal epitopeof the β-amyloid peptide was shown to bind the 2-13 peptide (SEQ IDNo:44) with an apparent KD of 7 nM. 2E7 did not bind to a selection ofpeptides in set (A) from mid regions of the β-amyloid peptide. In aseparate experiment the β-amyloid 1-40 peptide was captured via itsN-terminal aspartic acid residue that had been biotinylated. Thispeptide was captured onto a Biacore™ streptavidin coated chip aspreviously described. Antibody 2E7 injected at 66 nM for 1 minute couldnot bind this peptide. Therefore, it is concluded that the previouslydescribed N-terminal binding site was masked by the linker and capturemethod, thus further confirming the extreme N-terminus as containing thecore binding site

Binding to Cell Expressed Amyloid Precursor Protein (APP)

β-Amyloid is composed of peptides formed by proteolytic cleavage of atype I transmembrane precursor protein named amyloid precursor protein(APP). As APP has a large extracellular domain, binding to this proteincould potentially initiate an antibody-dependent cellular cytotoxicityreaction (ADCC).

To characterise binding of antibody to cell-surface full length APP anFMAT™ AB18200 based assay was utilised.

Transfection of HEK293T Cells with Wild Type APP DNA

HEK293T cells are maintained in DMEM F12 medium containing 10% (v/v) FCSand 1× Glutamax. Cells are seeded in 75 cm² tissue culture flasks andgrown to 60-80% confluency (18-24 h) prior to transfection. Fortransfection, 9 ug of DNA, (either wild type APP DNA (in PcDNA3.1(Invitrogen) vector), or vector only controls), is mixed with 0.3 ml ofOpti-MEM™ media. 30 ul Lipofectamine™ transfection agent is mixed with0.3 ml Opti-MEM™ media and the two mixtures pooled. The pooled mixturesare incubated at room temperature for 30 min prior to the addition of afurther 4.8 ml of Opti-MEM™ media. The final mixture is added to thecells (post washing with Opti-MEM™ media) for 5 h and 6 ml of 10% (v/v)newborn calf serum in DMEM is then added. 48 hrs post transfection,supernatant is removed and the monolayer washed in versene, and then 3ml of Versene™ chelating agent is added to each flask, incubated for 5mins at 37 C, and the detached cells pelleted at 200 g for 5 mins. Theresultant cell pellet is gently resuspended in 1 ml of assay buffer (2%heat treated serum, 0.5% BSA, 0.1% NaN₃ in PBS pH7.4, filtered through a0.2 um filter) to create a single cell suspension.

FMAT™ AB18200 Based Assay

Test antibodies (2E7, LN27 (Zymed) mouse IgG to extracellular domain ofAPP as a positive control, and an antibody G210 which recognises thex-40 form of the β-amyloid peptide as a negative control) were dilutedto 10 μg/ml in sterile filtered assay buffer (2% heat treat serum, 0.5%BSA, 0.1% NaN₃ in PBS pH7.2) in a polypropylene plate, and then afurther six serial 1:1 dilutions were performed down the plate. Assaybuffer only was used as a blank. 50 ul of a suspension of HEK293T cellstransfected with wild type APP DNA (Experiment 1: 10,000 cells;Experiment 2: 20,000 cells) was added to each well of a 96 well plate,to which 5 ul of each of the antibody solutions were added to duplicatewells. 50 ul/well of F-MAT™ blue anti mouse IgG stock, (antibody islabelled using F-MAT™ blue monofunctional reactive dye kit from ABI,4328408), diluted 1:500 (Experiment 1) and 1:1000 (Experiment 2) inassay buffer, was then added to each well and the plate briefly shakenand left to settle for 1 hr. The plate was then read using the ABI 8200fluorescence macro confocal cellular detection system (AppliedBiosystems).

Derived counts data were then interpreted using Excel™ spreadsheetsoftware. Briefly, mock transfected counts were subtracted from the fulllength APP transfected cell counts to obtain a specific signal for eachantibody. Two antibody concentrations that were on the linear part ofthe curve were chosen (1.25 and 0.63 ug/ml) and the background correctedderived counts at these concentrations expressed as the percentage ofthe LN27 antibody binding, and averaged over the two antibodyconcentrations. The resultant data is described in Table 3 (% of LN27binding ±SE)

Thus, within this assay system, the binding of 2E7 to cell surface APPis indistinguishable from that of the negative control antibody G210.

TABLE 3 antibody Experiment 1 Experiment 2 LN27 100.0 ± 7.1  100.0 ±4.7  G210 5.5 ± 1.3 2.0 ± 1.6 2E7 9.9 ± 3.7 2.2 ± 1.4

Binding to Amyloid Precursor Protein Derived Peptide

The previously described epitope mapping studies have shown thatantibody 2E7 binds to the extreme N-terminus of the β-amyloid peptide,with the aspartic acid residue at position 1 being essential forbinding. This suggests that the antibody recognises a ‘neo’ epitopeformed by cleavage of APP at the β-secretase site. This observationwould suggest that antibody 2E7 should not recognise adjacent APPpeptide sequence. To test this hypothesis an APP peptide (Peptide APP1,SEQ ID No:16) was synthesised which included residues 1-7 of theβ-amyloid peptide and the five adjacent APP derived amino acids. Thuspeptide APP1 contained contiguous amino acids from position 5 N-terminalto the BACE-1 cleavage site to position 7 C-terminal to the BACE-1cleavage site and was N-terminally acetylated. The ability of antibody2E7 to bind to the APP derived peptide APP1 and the β-amyloid 1-12peptide (peptide Aβ1) was compared using Biacore™ methodology (aspreviously described for epitope mapping). Antibody 2E7 showed highaffinity binding to the β-amyloid peptide Aβ-1, which contains the basicepitope 1-7. However, no binding was observed to the APP1 peptide whichalso contains the basic β-amyloid derived sequence 1-7.

Peptide Aβ1 SEQ ID No: 15 DAEFRHDSGYEVGSGK-biotin APP1 SEQ ID No: 16AcNH-SEVKMDAEFRHDGSGK-biotin

A combination of FMAT™ based cellular binding and Biacore™ based peptidemapping has been utilised to show that, in these formats, 2E7 has nobinding affinity for the full length APP protein. Given that theaspartic acid residue at position 1 of the β-amyloid peptide is requiredfor binding, it is concluded that 2E7 only recognises the ‘neo’N-terminus of β-amyloid and hence should not bind cell surface expressedAPP.

In vivo Biological Activity

I¹²⁵ β-Amyloid Efflux Model

A number of published studies have shown that β-amyloid antibodies canform complexes with β-amyloid peptide in the bloodstream. It is arguedthat this sequestration of peripheral β-amyloid allows for furtherefflux of CNS amyloid into the bloodstream (DeMattos RB, PNAS (2001),98(15); 8850-8855). An acute pharmacodynamic model was developed toscreen antibodies for their ability to complex with brain derivedβ-amyloid peptide in the bloodstream.

Anaesthesia (4% isoflurane) was induced in male C57/BL6J mice andmaintained (1.5% isoflurane) in 100% oxygen. Animals were then placed ina stereotaxic frame. Following midline incision along the sagittalsuture a bore hole was drilled through the skull and a guide cannula wasinserted into the lateral cerebral ventricle (co-ordinatesanterioposterior (AP)-0.5 mm, lateral (L)+0.7 mm, ventral (V)-2.5 mm). Afurther two bore holes were drilled through the skull into whichcortical screws were placed. The cannula was anchored in place bycyanoacrylate gel and the incision was sutured around the cyanoacrylategel headcap. Post-operatively the mice received 0.3 ml salinesubcutaneously and were placed in a warm environment to recover fromanaesthesia. On recovery of the righting reflex, mice were housed singlyand received 5 days standard post-op care. No procedures were permittedfor a further 5 days or until pre-operative body weight was regained.Following recovery, cannula placement was verified by the angiotensin IIdrinking response. Each mouse received an intracerebroventricular (ICV)administration (50) of 100 ng angiotensin II (All) (made up in 0.9%saline). Following administration of All, water intake was observed for15 minutes. Mice with a positive dipsogenic response to All (sustaineddrinking) were included in the studies, which commenced no sooner thanfive days post All injection.

On the day of study the mice were placed for 5-10 minutes in a warmenvironment to induce vasodilation, necessary for ease of injection intothe tail vein. Test antibody (600′4) or PBS vehicle (dose volume nogreater than 10 ml per kg body weight) was injected via the tail veinand mice were returned to their individual cages post-injection. Atexactly one hour post tail vein injection, mice were slowly ICV injected(20 per minute) with 2 ng (1 μCi) of I¹²⁵ beta-amyloid 1-40 (AmershamBiosciences, UK) in a dose volume of 50. At exactly four hours post ICVdose, 500 of trunk blood was collected and the radioactivity levelmeasured on a scintillation counter.

Mice that had been injected into the tail-vein with 2E7 (n=6 pertreatment group) showed a statistically significant increase in theradioactive signal (counts per minute—CPM) in 500 of trunk bloodcompared with the CPM signal detected in vehicle injectedmice—(CPM—vehicle: 1339.7±496.2 vs. 2E7 4387.9±980.3;ANOVA:F(2,13)=4.97, p<0.05. Post-hoc LSD: p=0.01 2E7 vs. vehicle[post-hoc Duncans: p=0.02 2E7 vs, vehicle]).

In two further studies with 2E7 conducted with the identical protocol,similar increases in amyloid efflux into blood when compared withvehicle injected controls were observed (CPM blood: Vehicle 352+/−113versus 2E7 2397+/−353, and Vehicle 1281+/−312 versus 2E7 5291+/−885;ANOVA with post-hoc LSD test p<0.001 vs. vehicle).

Transgenic CNS R-Amyloid Lowering Models

1. β-Amyloid Load following 4 week dosing of 2 month old TASTPM mice

Male and female TASTPM transgenic mice (double-mutant APPswe×PS1.M146V,Howlett DR (2004) Brain Research 1017 (1-2) 130-136) aged between 61 and65 days at the start of the study and were singly housed. Equal numbersof mice were assigned to each treatment group (N=12 per group) and wererandomized according to gender and age. Treatment groups comprised thefollowing: A: MOPC21 (antibody with unknown specificity, Holton et al(1987) J. Immunol 139(9) 3041-3049, negative control), B: 2E7 (testantibody). All antibodies were dissolved in PBS and were dosed by theintraperitoneal route. Irrespective of animal weight, 300 ug of antibodywas administered. Animals were dosed twice weekly for four weeks. Oneday after the final dose, animals were euthanased by overdose withsodium pentobarbital. Brains were dissected and hemisected. Hemisectedbrain samples were collected into pre-weighed 2 ml Eppendorf™ tubes andsnap frozen. Samples were subsequently thawed, reweighed and 1 ml of 5Mguanidine HCl containing Complete protease Inhibitor™ tablets(Boehringer Mannheim) added, before the samples were homogenized andincubated at 4° C. for >90 min with constant agitation.

Samples were then diluted 1 in 10 into assay buffer (50 mM Tris HCl,pH7.4, 150 mM NaCl, 0.05% Tween-20+1% BSA), vortexed and spun at 20,000G for 20 mins at 4° C. The supernatant was removed and added astriplicate samples to the assay plate.

The levels of Aβ40 and Aβ42 were measured using a sensitive plate basedelectrochemiluminescent immunoassay (BioVeris™) employing C-terminalspecific β-amyloid antibodies (to Aβ40 or Aβ42) labelled with Oritag™specific label to facilitate detection (BioVeris™) used to captureeither Aβ40 or Aβ42, along with a biotinylated N-terminal specific Aβantibody. Antibody-Aβ complexes were captured with streptavidin coatedbeads that bind biotinylated antibodies (Dynabeads™, Dynal) incubatedovernight at room temperature with vigorous mixing and assayed in aBioVeris™ M8 photodetector. Standard curves were constructed using humanAβ40 and Aβ42 peptides in assay buffer containing the requiredconcentration of Guanidine HCl. Data was analysed using Excel Robosage™statistical analysis software and Aβ levels expressed as pmole/g tissue.

In this paradigm, treatment with 2E7 antibody reduced CNS Aβ42 load by37% (p<0.001). and CNS Aβ40 by 23% (p<0.001).

In subsequent studies under similar experimental conditions, 2E7antibody reduced CNS Aβ42 load by 38% (Study 1, males only), 22% (Study2, non-significant) and 39% (Study 3, males, p=0.001) and 13% (Study 3,females, non-significant) when compared to PBS treated animals. In thesestudies 2E7 also reduced CNS Aβ40 by 18% (Study 3, males, p=0.017) andoffered a non-significant reduction in CNS Aβ40 by 25% (Study 1, malesonly), <1% (Study 2) and a non-significant increase of 3% (Study 3,females) when compared to PBS treated animals.

2. β-Amyloid Load Following 4 Month Dosing of 4 Month Old TASTPM Mice

Briefly, 4 month old TASTPM transgenic mice were dosed 300′4 of antibodyonce or twice weekly via an intraperitonial (i.p.) route. After 4 monthsof dosing CNS β-amyloid levels were measured by ELISA and plaque loadwas measured by immunohistochemistry. Between the ages of 4 and 8months, the CNS β-amyloid load increases exponentially and consequently,plaque pathology rapidly develops (Howlett DR (2004) Brain Research 1017(1-2) 130-136).

Mice were aged between 120 and 128 days at the start of the study andwere singly housed. Similar numbers of mice were assigned to eachtreatment group (N=20 or 21 per group) and were randomized according togender and age. Treatment groups comprised the following: A: PBS(vehicle) dosed twice weekly, B: 2E7 dosed once weekly, C: 2E7 dosedtwice weekly, D: PBS dosed once weekly. A 300 microgram dose (79microlitres volume) of 2E7 was administered via the intraperitonealroute. Vehicle treated animals received the same volume of PBS. Animalswere dosed for eighteen weeks. TASTPM mice are liable to sufferspontaneous seizures and as a result a number of animals died during thecourse of the study. Final numbers were as follows A: 4 females, 9males; B: 5 females, 8 males; C: 4 females, 9 males; D: 2 females, 9males. Two or four days after the final dose (equal numbers per group)animals were euthanased by overdose with sodium pentobarbital. A tailtip sample from each mouse was taken for confirmation of the genotype.Brains were dissected and hemisected. The right hemisphere was fixed byimmersion in 4% paraformaldehyde and processed for histology. The lefthemisphere was collected into pre-weighed 2 ml Eppendorf™ tubes, frozenon dry ice and stored at −80° C. for subsequent analysis of amyloidcontent. Prior to analysis, samples were thawed, reweighed and 1 ml of5M guanidine HCl containing Complete protease Inhibitor™ tablets(Boehringer Mannheim) added, before the samples were homogenized andincubated at 4° C. for >90 min with constant agitation.

Samples were then diluted 1 in 10 into assay buffer (50 mM Tris HCl,pH7.4, 150 mM NaCl, 0.05% Tween-20+1% BSA), vortexed and spun at 20,000G for 20 mins at 4° C. The supernatants were diluted a further 1:1000and added as triplicate samples to the assay plate.

The levels of Aβ40 and Aβ42 were measured as for the 4 week dosingstudy.

An analysis of variance was used with treatment, sex and dosing scheduleincluded in the model as fixed effects. All of the interactions betweenthe three factors were also included. There were no significantdifferences between the two dosing schedules (once or twice weekly).With this experimental design, firstly it could be assessed if therewere any significant differences between the dosing schedules andsecondly, as there were no such significant differences, data from thetwo dosing schedules could be combined, thus increasing the power of theexperiment by doubling the number of mice in the analysis.

In this paradigm, treatment with 2E7 antibody reduced CNS Aβ42 load by22.5% (p=0.0152). Levels of CNS Aβ40 were also lowered by 12.1%, butthis figure did not reach statistical significance (p=0.118).

A complex immunohistochemical analysis of these samples was performed todefine the area of brain tissue showing plaque pathology. Sections weretaken from the cortex at the level of the caudate and from the cortex atthe level of the hippocampus. Adjacent sections were stained with eitheran Aβ40 or Aβ42 specific antibody or alternatively with the amyloidstain Congo Red. Using image analysis software, the area of the sectionstained for plaque was expressed as a percentage of the total sectionarea.

After fixation, the PFA-immersed half brains were coronally cut in abrain matrix into 6×2 mm thick sections. These 2 mm sections will bereferred to as sections A to F, A being the most rostral and F the mostcaudal. Sections A, B & C and D, E & F were placed in separate embeddingcassettes numbered for each animal. Cassettes were held in PFA untilready for processing and embedding.

Embedding was undertaken on a Citadel™ 1000 (Shandon) tissue processor.All tissues received the following processing regimen:—

70% IMS—1 hr 100% IMS—3×1 hr

100% ethanol—2 hr100% isobutyl alcohol; 1×2 hr; 1×1.5 hr

Histoclear™—2×1.5 hr

Paraffin wax—2×2 hr

On completion of the processing cycle, the wax impregnated tissuesections were transferred to molten-paraffin wax filled base moulds andembedded utilising a Histocentre™ (Shandon) paraffin embedding system.Tissue was embedded such that sections A, B & C went into one mould; D,E & F into a second mould. This was carried out for all sets of sectionsie. each hemisected brain resulted in two wax blocks of three sectionseach. Sections were placed in the moulds such that the caudal surface ofeach piece became the future cutting surface. Care was taken to ensurethat each section was pushed well down in the mould so that microtomingof each would occur in parallel. The perforated processing cassette wasthen carefully placed onto each mould which was then topped up withmolten wax. Embedded blocks were then cooled on the refrigerated plateuntil they could be removed from the moulds. Blocks were stored at roomtemperature until required for microtoming. Blocks were cut at randomand 5 micron sections floated onto prelabelled gelatine coated slides(Superfrost™, Erie Scientific Company) slides. Two sections were floatedonto each slide. Wherever possible, consecutive sections were mountedand slides were numbered consecutively from 1 to 25. Fifty sections (25slides) were taken from each block. Slides were dried on a hot plate andthen stored at room temperature until required.

Immunohistochemistry was undertaken on sets of 30 slides. On each slide,the top section was labelled with an Aβ40 antibody (G30, rabbitpolyclonal recognising x-40 β-amyloid), the lower section with the Aβ42antibody, 20G10, monoclonal antibody recognising x-42 β-amyloid. Aminimum of 5 sections per antibody per block were labelled.

Labelling was carried out as follows. Following dewaxing throughHistoclear and graded alcohols, sections were immersed in 85% formicacid for 8 minutes and then blocked in 0.3% hydrogen peroxide for 30minutes to block endogenous peroxidases. Antibodies G30 and 20G10 wereboth applied overnight at 1:1000 dilutions, sections being left at 4° C.Development of the sections was with the respective biotinylated antirabbit and anti-mouse secondaries. Colour development was accomplishedwith a diaminobenzidine tetrahydrochloride staining kit (DAB™, VectorLabs). Sections were briefly counterstained with Mayer's hematoxylinbefore being dehydrated, cleared and cover-slipped.

Sections were left to dry for at least 48 hours before microscopy.Images were captured on a Leica DMRB™ microscope equipped with digitalcamera. Images were analysed using Qwin™ software (Leica) and resultspresented as % of the section area that was labelled by the Aβ antibody.

An analysis of variance was used with treatment, sex and dosing scheduleincluded in the model as fixed effects. All of the interactions betweenthe three factors were also included. There were no significantdifferences between the two dosing schedules (once or twice weekly).With this experimental design, firstly we could assess if there were anysignificant differences between the dosing schedules and secondly, asthere were no such significant differences, data from the two dosingschedules could be combined, thus increasing the power of the experimentby doubling the number of mice in the analysis.

In this paradigm, treatment with 2E7 antibody reduced plaque pathologyas measured with an antibody recognising Aβ42. Plaque pathology wasreduced by 27.1% (p=0.0026) in the cortex at the level of thehippocampus and 43% (p<0.0001) in the cortex at the level of thecaudate. Plaque pathology was also reduced when measured with anantibody recognising Aβ40. Plaque pathology was reduced by 16.6%(p=0.0421) in the cortex at the level of the hippocampus and 17.3%(p=0.0342) in the cortex at the level of the caudate.

No evidence of microhaemorrhage (as determined by Perls' Prussian Blue)was observed in any mice from this study treated with vehicle or 2E7.This method visualises ferric iron (iron is an essential constituent ofthe oxygen-carrying haemoglobin found in red cells) by producing aninsoluble blue compound. All levels of brain from all animals wereclear.

Cognition Models

Following the 4 month dosing of 4 month old TASTPM mice as describedabove, these mice were tested in two models of cognition: the objectrecognition assay and the fear conditioning assay.

Object Recognition Assay

The object recognition assay exploits the animals' natural propensity toexplore novel objects and relies on the animals' ability to recall anobject which had been explored previously (familiar object). Eight monthold TASTPM mice have been reported to demonstrate a deficit in theability to distinguish between novel and familiar objects (Howlett etal., 2004) indicating impaired cognitive performance in these animals.In this study, however, 8 month-old TASTPM mice treated with vehiclefailed to demonstrate cognitive impairment i.e. they were able todistinguish between novel and familiar objects. There was therefore nowindow to investigate any potential therapeutic effect resulting fromtreatment with 2E7.

Fear Conditioning Assay

The fear conditioning model was designed to test the animals' ability tocorrelate a previous painful stimulus with a contextual or cued signaland recall this when presented with the same context or tone followingXh delay. In this study 8-month old TASTPM mice treated with vehicle(once or twice weekly) exhibited a deficit in contextual differentiationindicative of cognitive impairment in these animals. This deficit wasunaffected by treatment with 2E7 when administered once of twice weekly

4 Month Dosing of 6 Month Old TASTPM Mice

This study involved the administration of 2E7 (300 ug i.p. twice weekly)to TASTPM mice for 4 months, starting at 3 months of age. Controlanimals received IgG2A in PBS. As described above, brains were dissectedand hemisected. The right hemisphere was fixed by immersion in 4%paraformaldehyde and processed for histology. The left hemisphere wascollected into pre-weighed 2 ml Eppendorf™ tubes, frozen on dry ice andstored at −80° C. for subsequent analysis of amyloid content.

A preliminary analysis of a single section from each of a randomselection of brain samples (n=6 vehicle, n=7 2E7 treated group) by IHCwas undertaken using the same general protocol as above. Statisticalanalysis (Student's t-test) shows that there was a significant decreasein Aβ42 plaque load in thalamus (71.9%, p=0.007) and inthalamus+cortex+hippocampus (54.1%, p=0.022) in mice dosed with 2E7 butno significant change in Aβ40.

For biochemical measurement of brain Aβ40 and Aβ42, samples wereprocessed and measured as above (dilution factor 1:10,000). Aβ42 wassignificantly decreased (p=0.01) by 29.9% in mice dosed with 2E7 (n=12control, n=16 treated). Aβ40 concentrations were also decreased (22.6%)but this decrease failed to reach statistical significance (p=0.052).

Cloning of Hybridoma Variable Regions Variable Region Sequences

Total RNA was extracted from 2E7 hybridoma cells and heavy and lightvariable domain cDNA sequences were then generated by reversetranscription and polymerase chain reaction (RT-PCR). The forward primerfor RT-PCR was a mixture of degenerate primers specific for murineimmunoglobulin gene leader-sequences and the reverse primer was specificfor the antibody constant regions, in this case murine isotype IgG2a forthe heavy chain and murine kappa for the light chain. Primers weredesigned according to the strategy described by Jones and Bendig(Bio/Technology 9:88, 1991). RT-PCR was carried out in duplicate forboth V-region sequences to enable subsequent verification of the correctV-region sequences. The V-region products generated by RT-PCR werecloned (Invitrogen TA Cloning Kit) and sequence data obtained.

2E7 V_(H) Amino Acid Sequence (SEQ ID No: 17)EVKLVESGGGLVQPGGSLKLSCAVSGFTFSDNGMAWVRQAPRKGPEWIAFISNLAYSIDYADTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCVS GTWFAYWGQGTLVTVSA2E7 V_(H) DNA Sequence (SEQ ID No: 18)GAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCACTTTCAGTGACAACGGAATGGCGTGGGTTCGACAGGCTCCAAGGAAGGGGCCTGAGTGGATAGCGTTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCTAGAGATAATGCCAAGAATACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTACTATTGTGTAAGCGGGACCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTG CA2E7 V_(L) Amino Acid Sequence (SEQ ID No: 19)DVVLTQTPLSLPVSLGDQASISCRVSQSLLHSNGYTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTRH VPYTFGGGTKLEIK2E7 V_(L) DNA Sequence ((SEQ ID No: 20)GATGTTGTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGAGTTAGTCAGAGCCTTTTACACAGTAATGGATACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAACTAGACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

Complementarity Determining Regions (CDRs) are underlined in the aminoacid sequences.

Cloning and Expression of 2E7 Chimera

A chimeric 2E7 antibody (2E7c) consisting of the parent murine V regionsgrafted on to human IgG1 (Fc mutated (L235A, G237A)) for the heavy chainor human C kappa regions for the light chain was generated in order toexpress recombinant antibody material that could be used to confirm thecorrect cloning of functional murine V regions. DNA encoding 2E7 murineheavy and light chain V regions and endogenous murine signal sequenceswas cloned in frame into the mammalian expression vectors RLD-bshe (forthe heavy chain) and RLN-bshe (for the light chain) already containinghuman constant regions (IgG1 Fc mutated (L235A, G237A) or human C kappa,respectively).

Elements of RLD-bshe Expression Vector for Heavy Chain Expression:

Base Pairs Description of DNA segment   0-1014 Promoter (SV40/RSV)1015-2442 Antibody heavy chain 2443-2765 Poly A 2766-3142 BG Promoter3239-3802 DHFR 3803-4162 Poly A 4163-6322 Total backbone 5077-5937 Betalactamase (complementary strand)(position of elements and overall size of vector given above are forillustration purposes only and will depend upon the size of the antibodychain insert)

Elements of RLN-bshe Expression Vector for Light Chain Expression:

Base Pairs Description of DNA segment   0-1014 Promoter (SV40/RSV)1015-1731 Antibody light chain 1732-2051 Poly A 2388-2764 BG Promoter2774-3568 Neomycin 3569-3876 Poly A 3877-6063 Total backbone 5077-5937Beta lactamase (complementary strand)(position of elements and overall size of vector given above are forillustration purposes only and will depend upon the size of the antibodychain insert)

Clones with correctly cloned V_(H) and V_(L) sequences were identifiedand plasmids prepared for expression in suspension culture CHO cells.Expressed 2E7c antibody was purified from cell culture supernatant byprotein A chromatography on a FPLC system, and then tested for bindingto Aβ by ELISA and SPR using Biacore™ technology. The results indicatedthat the correct 2E7 mouse V regions were cloned and expressed,resulting in a functional antibody with similar characteristics to theparent murine antibody 2E7.

Light Chain Humanisation

A human acceptor sequence with the Genpept ID CAA51135 (SEQ ID No:24)and Genbank Accession No X72467, which had 77% identity on the aminoacid level (including CDRs) was selected as the acceptor framework.Construct L1 is a graft of the murine CDRs from the 2E7 VL domain intothis acceptor framework.

Heavy Chain Humanisation

Human sequence Genbank accession No M99675 (SEQ ID No:21) an allele ofthe VH3-48 gene with 74% identity on the amino acid level (includingCDRs 1 and 2) to the 2E7 mouse variable heavy region was selected as thehuman heavy chain acceptor framework together with the human JH4minigene. Three humanised variable heavy chain variants were designedbased on the M99675 sequence and JH4. H1 is a graft of the murine CDRsusing the Kabat definition with two additional framework back mutationsat positions 93 and 94. H2 and H3 were both derived from H1, butincorporated one additional framework mutation which were different ineach construct; (positions 24 and 48 respectively; see Table 4).

TABLE 4 Template Residue Construct frameworks (Kabat#) Human Mouse H1M99675 93, 94 A and R V and S and JH4 respectively respectively H2 H1 24A V H3 H1 48 V I

Construction of Humanised Heavy and Light Chain DNA

Humanised V regions were synthesised de novo by build up of overlappingoligos and PCR amplification. Restriction sites for cloning intomammalian expression vectors RLD-bshe and RLN-bshe and humanimmunoglobulin signal sequences derived from the chosen human acceptorframeworks were included. The DNAs encoding the humanised V regions (H1(SEQ ID NO:27), H2 (SEQ ID NO:29), H3 (SEQ ID NO:31), L1 (SEQ ID NO:33))together with signal sequences and restriction sites were then cloned inframe into mammalian expression vectors: H1, H2 and H3 into RLD-bshe togenerate DNA encoding three full length human IgG1 Fc mutated heavychains each containing mutations L235A and G237A, full length H1 (SEQ IDNO:35), full length H2 (SEQ ID NO:37) and full length H3 (SEQ ID NO:39);L1 was cloned in frame into RLN-bshe containing the DNA encoding humankappa constant region to generate DNA encoding a full length human kappalight chain (SEQ ID NO:41).

Representative Examples of Expression of Humanised Heavy and Light ChainAntibody Combinations

CHOK1 cells were transiently transfected at small scale with allcombinations of humanised light and heavy chain DNA constructs: L1+H1,L1+H2, L1+H3 (SEQ ID Nos: 35+41, 37+41, 39+41) in 6-well plates. CHOK1cells passaged in DMEM F12, with 5% ultra low IgG foetal bovine serumand 2 mM glutamine were grown to confluency in 6-well plates. Theconfluent cells were transfected with a total of 7.5 μg DNA: 30 μgTransfast lipid (Promega) in Optimem Glutamax medium (Invitrogen).Transfected cells were incubated at 37° C. with 5% CO₂. At 72 hourssupernatants were harvested and assayed for antibody concentration andthen tested for binding to human Aβ by ELISA. Humanized L1 combined withthe three humanized heavy chains all expressed complete antibody thatbound to human Aβ.

Humanized antibodies were also expressed in large scale transient CHOK1cell transfections using liposomal delivery of DNA (eg TransFast(Promega)) and expression in culture bottles. For optimization ofexpression levels in transient transfections a heavy to light chainexpression vector DNA ratio of 1:6 was used. Material from transienttransfection was purified using ProSepA columns or FPLC with ProSepAHiTrap columns.

Assessment of 2E7 Humanised Variants H1L1, H2L1 and H3 L1 in B-AmyloidBinding ELISA

2E7 H1L1, H2L1 and H3L1 humanised variants were assessed for binding tohuman Aβ peptide (1-40) biotinylated at the C terminus. The chimeric 2E7was used as a reference. Tables 5-7 show results with various batches ofpurified material from large scale transient transfections.

TABLE 5 EC₅₀ Standard ELISA MAb (μg/ml) Error Aβ binding 2E7c Chimera0.033 0.00144 H1L1 0.035 0.00142 H2L1 0.048 0.00202 H3L1 0.044 0.00105

TABLE 6 EC₅₀ Standard ELISA MAb (μg/ml) Error Aβ binding 2E7c Chimera0.043 0.00183 H1L1 0.051 0.00164 H2L1 0.044 0.00191 H3L1 0.055 0.00094

TABLE 7 EC₅₀ Standard ELISA MAb (μg/ml) Error Aβ binding 2E7c Chimera0.044 0.00169 H1L1 0.047 0.00265 H2L1 0.041 0.00174 H3L1 0.040 0.00116

These results indicated very similar Aβ binding profiles for each of the2E7-derived humanised variants. Comparison of the EC50 values to the2E7c showed little loss of Aβ binding activity had been incurred throughthe humanization process.

Comparison of 2E7 Humanised Variants by Competition ELISA

E7c chimeric and humanised antibodies H1L1, H2L1 and H3L1 were assessedfor their ability to inhibit the binding between the human Aβ peptideand the parental mouse 2E7 MAb in a competition ELISA.

Two types of competition ELISA were established in order to compare theAβ binding activity of the three humanised variants compared to the 2E7chimeric antibody.

1) Immobilised β-amyloid; biotinylated human Aβ peptide (1-40) wasimmobilized through Streptavidin on ELISA plates Mouse 2E7 antibody wasadded at a constant concentration along with a dilution series of2E7-derived humanised variant antibodies. Bound mouse 2E7 MAb was thendetected with anti-mouse IgG conjugate. Table 8 shows results of twoassays.

TABLE 8 Experiment Experiment 1 Standard 2 Standard Competitor MAb IC₅₀(μg/ml) Error IC₅₀ (μg/ml) Error 2E7c Chimera 1.31 0.20 1.29 0.13 H1L11.62 0.40 1.76 0.21 H2L1 1.28 0.26 1.66 0.28 H3L1 1.53 0.16 1.39 0.232)β-amyloid in solution; a constant concentration of β-amyloid waspre-incubated with a dilution series of humanised 2E7 antibodyvariants—the mixture including complexed and free amyloid was added fora short time to wells containing immobilised mouse 2E7 MAb. The amountof free β-amyloid that was still available for binding the immobilisedparental 2E7 MAb was then detected. Table 9 shows results of two assays.

TABLE 9 Experiment Experiment 1 Standard 2 Standard Competitor MAb IC₅₀(μg/ml) Error IC₅₀ (μg/ml) Error 2E7c Chimera 0.052 0.006 — — H1L1 0.1140.014 0.140 0.024 H2L1 0.075 0.009 0.119 0.014 H3L1 0.069 0.004 0.1150.013

All humanised antibody variants inhibited the binding of mouse 2E7 MAbto β-amyloid with a very similar profile. IC₅₀ values generated for H2L1and H3L1 variants were consistently close to that of the 2E7c chimera(where used), which had the highest inhibitory activity in both assays.However, variant H1L1 showed a somewhat reduced inhibitory activity inboth assays, indicating a possible slightly lower affinity forβ-amyloid.

SPR Biacore™ Analysis of 2E7, 2E7c, H1L1, H2L1, H3L1

The kinetics parameters of recombinant mouse 2E7 MAb, chimeric 2E7c andhumanized variants H1L1, H2L1 and H3L1 binding to human beta-amyloidpeptide (1-40) and (1-42) were assessed using Biacore™ analysis on aBiacore™ 3000. Two different assay formats were used.

Method A

(i) Briefly, <20 resonance units of beta-amyloid 1-40 peptide(biotinylated at the C-terminus) were captured on a streptavidinbiosensor chip (as used for Table 10A). The antibodies were diluted downin HBS-EP buffer and passed over the streptavidin/beta-amyloid surfaceat concentrations ranging from 0.001 nM-8 nM (for Table 10A). Twoseparate runs were carried out; each run was carried out on a newstreptavidin/beta-amyloid surface. Runs 1 and 2 were essentially thesame though there were some differences in the parameters used; Run 1was carried out using a chip surface on which 16 RU's of beta-amyloidwere captured, and antibody concentrations of 0.001 nM-8 nM were used,an association time of 4 minutes and a dissociation time of 20 minuteswere used at a flow rate of 500 per minute. For Run 2, less than 10 RU'sof beta-amyloid were captured and antibody concentrations of 0.003125nM-8 nM were used. The flow rate and association times were the same asRun 1, however the dissociation time was reduced to 15 minutes.(ii) Beta amyloid (1-40) and (1-42) were amine-coupled on differentsurfaces of a CM5 biosensor chip to a level of <20 resonance units (asused for Table 10B). The antibodies were diluted down in HBS-EP bufferand passed over the biosensor/beta-amyloid surface at concentrationsranging from 1 nM-64 nM (as used for Table 10B).

Method B

In the second instance the assay was reversed, in that antibodies werefirst captured to a level of 1000-2500 resonance units on an anti-mouseIgG polyclonal antibody surface (for recombinant mouse 2E7 MAb) or aprotein A surface (for humanized H2L1) of a CM5 biosensor chip. Freshlyprepared beta-amyloid (1-40) or (1-42) was diluted down in HBS-EP bufferand passed over the captured-antibody surface at concentrations rangingfrom 4-500 nM (Table 10C and 10D).

In both methods, regeneration was via a pulse of 100 mM H₃PO₄, and forTable 10A data also followed by a pulse of 50 mM NaOH. The surface wasshown to be stable and unaffected by regeneration. All runs were doublereferenced against buffer blank injections. Analysis was carried outusing the Biacore™ analysis software BIAevaluation version 4.1.

Results

Method A(i) was used to rank order the antibodies by beta-amyloidbinding kinetic data. The data obtained is shown in Table 10A. Thisshows that the parental 2E7 Mab has a KD of 36.1 μM forstreptavidin-captured beta-amyloid. The chimeric mouse-human antibodyshowed a slightly reduced KD of 45.8 μM and the humanised constructsrange from 54 (H2L1) to 93.6 μM (H1L1). In conclusion this demonstratesthat the humanisation procedure had been very successful and very littleaffinity had been lost. The additional backmutations introduced for H2and H3 had a small but beneficial effect, although the differencesbetween H2 and H3 constructs are within the standard deviations forthese experiments.

TABLE 10A Antibody ka kd KD(pm) 2E7 Run 1 1.61e6 6.17e−5 38.3 Run 21.69e6 5.72e−5 33.8 Average(SD) 1.65e6 5.97e−5 36.1(3.2) c2E7 Run 11.34e6 6.44e−5 48.1 Run 2  1.3e6 5.65e−5 43.5 Average(SD) 1.32e6 6.10e−545.8(3.3) H1L1 Run 1 5.60e5 5.32e−5 95.0 Run 2 6.37e5 5.87e−5 92.2Average(SD) 5.99e5 5.60e−5 93.6(2.0) H2L1 Run 1 9.91e5 5.76e−5 58.1 Run2  1.1e6 5.49e−5 49.8 Average(SD) 1.05e6 5.63e−5 54.0(5.9) H3L1 Run 18.24e5 6.26e−5 76.0 Run 2  8.3e5 4.75e−5 57.2 Average(SD) 8.27e5 5.47e−566.6(13.3)

Method A(ii) was used to confirm that the additional two amino-acidresidues on the C-terminus of beta-amyloid (1-42) compared tobeta-amyloid (1-40) did not significantly alter the binding propertiesof 2E7 and H2L1. The data obtained is shown in Table 10B and did confirmthis.

TABLE 10B Beta- ka kd KD Antibody amyloid (Ms⁻¹) (s⁻¹) (pM) 2E7 1-404.05e5 1.28e−4 317 1-42 3.82e5 1.51e−4 394 H2L1 1-40 3.33e5 1.22e−4 3661-42 3.40e5 1.55e−4 456

Method B was used to negate avidity effects potentially seen in thefirst assay format. Avidity effects, caused by both Fab domains of asingle antibody molecule binding at the same time to two adjacentbeta-amyloid molecules on the biosensor surface (or in multimeric formsof beta-amyloid), would increase the apparent affinity of binding.Affinity measurements obtained using Method B are shown in Table 10C.

TABLE 10C KD (nM) ka kd With Standard Antibody (Ms⁻¹) (s⁻¹) Deviation n= 3 2E7 2.83e5 ± 0.54e5 4.28e−4 ± 0.65e−4 1.58 ± 0.55 H2L1 1.06e5 ±0.27e5 7.50e−4 ± 0.50   7.32 ± 1.64

Evidence that this assay provided true 1:1 binding affinities wasobtained when Fab fragments of H2L1, obtained by papain digestion, boundstreptavidin-captured beta-amyloid (1-40) by a similar method to MethodA(i) with an estimated KD of 2.4 nM.

Method B was also used to confirm that the additional two amino-acidresidues on the C-terminus of beta-amyloid (1-42) compared tobeta-amyloid (1-40) did not significantly alter the binding propertiesof an identical sequence clone to mouse 2E7 MAb, named 2F11. The dataobtained is shown in Table 10D.

TABLE 10D Beta- ka kd KD Antibody amyloid (Ms⁻¹) (s⁻¹) (nM) 2F11 1-422.39e5 2.74e−4 1.14 2F11 1-40 2.99e5 3.92e−4 1.31

In a study similar to the epitope mapping study on 2E7 using the SurfacePlasmon Resonance assay described above, H2L1 behaved similarly to 2E7in binding to the peptide encompassing amino acids 1-12 (Aβ1, SEQ IDNo:15) of the β-amyloid peptide and not to the peptide encompassingamino acids 2-13 of the β-amyloid peptide (Aβ2-13, SEQ ID No:44).

Activity of H2L1 in the I¹²⁵ β-Amyloid Efflux Model

In order to functionally compare the humanised H2L1 with the parentmouse monoclonal 2E7, both were tested on the same day in the I¹²⁵β-amyloid efflux model described above.

Both H2L1 and 2E7 significantly increased counts per minute (CPM) inblood compared with vehicle control. CPM of radioactivity in blood wasas follows (Vehicle: 1940±166; 2E7: 10065±1386; H2L1: 10913±1535).Statistics used were ANOVA with post-hoc LSD test. n=7 vehicle, n=6 2E7,n=6 H2L1, (p<0.001 for each test compound vs. vehicle).

This data provides further evidence that the humanised H2L1 antibody hasretained the functional properties shown with the mouse 2E7 molecule.

Investigation of the Pharmacokinetics of H2L1 and 2E7

The terminal half life of test antibody in mice was investigated. Testantibody was administered by a 1 h intravenous infusion to 4 mice toachieve a target dose of 400 ug per mouse. Serial blood samples weretaken from each mouse up to 5 days after dosing (one mouse from the 2E7group did not complete the study and one from the H2L1 group was removedfrom subsequent analysis because it became apparent the dose had notbeen administered i.v.). Antibody levels were measured using a β-amyloidcapture ELISA.

Analysis of the data indicates that the humanised antibody H2L1 has aterminal half life of circa 82 hours in mice (Table 11), which iscomparable to that of the parent mouse monoclonal antibody 2E7 (circa 75hours).

TABLE 11 Mean ± SD Parameter (n = 3) Cmax (ug/mL) 291 ± 43  Tmax (h) #2.0 (1.1-2.1) CLp (mL/h/kg) 0.9 ± 0.1 t½ (h) 82 ± 4  Vss (mL/kg) 94 ± 12# median and range Cmax Observed maximum plasma concentration. Tmax Timeof the observed maximum plasma concentration CLp Total plasma clearance;Dose/AUC_((0-inf)). t½ Terminal phase half-life was determined as theratio of In2/z where z is the terminal phase rate constant; calculatedusing unweighted linear regression analysis (after log transformation)on those concentration-time pairs occurring after the visually assessedonset of the terminal log-linear phase. Vss Volume of distribution atsteady-state; CLp x MRT_(0-inf).

Effect of H2L1 on Peripheral Amyloid Load in Aged Non-Human Primates

A study was conducted in aged Cynomolgus monkeys (approximately 15 yearsold) to investigate the exposure response relationship with respect toamyloid/H2L1 complex formation and clearance and the subsequent effectson CSF and CNS amyloid levels. Weekly lumbar CSF (taken under ketaminesedation) and blood samples were collected 3 weeks prior to 1^(st) doseof H2L1. Immediately following sampling on week 3, animals receivedplacebo (n=10), 0.1 mg/kg (n=5), 1 mg/kg (n=5) or 10 mg/kg (n=10) H2L1and then every 2 weeks for 12 weeks. Blood samples for plasma analysisof H2L1 and total Aβ₄₂ were taken weekly. CSF samples for quantificationof Aβ_(40/42) were collected every 2 weeks. Following completion of thedosing period, animals were euthanased for the purpose of brainquantification of beta-amyloid by biochemical analyses as describedabove and investigation of microhaemorrhage. In the lowest dose group(0.1 mg/kg), animals were euthanased in a staggered fashion to evaluatethe potential time course effect in brain levels as a consequence oftermination of dosing and hence saturation of the plasma amyloid pool.

This study was approved by the Institutional Animal Care and UseCommittee (IACUC) of MACCINE Pte Ltd, or “Maccine” prior to start of theexperimental phase. The IACUC protocol number was #08-2006. GSK hasperformed a site visit of Maccine and has reviewed their ethical reviewprocess and found it acceptable

Plasma samples were serially diluted 1:10 to 1:50000 and added to Aβ₄₀coated ELISA plates. Standard curves were created ranging from 0-10μg/ml H2L1 in diluent. Following an overnight incubation at 4° C. H2L1was visualised using anti-human IgG horseradish peroxidase(Amersham—diluted 1:2000 in diluent) and tetramethylbenzidene detectionsystem. Following single and repeat iv bolus administration, plasmalevels of H2L1 appeared to increase in a dose dependent fashion. Therewas no evidence of severe non-linearities in the pharmacokinetics,indicating that for the majority of the dosing interval, excess molarconcentrations of H2L1 in the plasma compared with free amyloid levelswere achieved.

Total Aβ₄₂ was measured in neat plasma using a commercially available Aβ1-42 ELISA kit (Innogenetics) in accordance with the manufacturersinstructions, with standard curves ranging from 500-7 μg/ml created inkit diluent. Samples and standards are incubated overnight at 4° C.before assaying in duplicate according to kit instructions. It should benoted that due to the interference of the detection antibody suppliedwith the Aβ₄₂ assay, this kit cannot be used to measure free Aβ₄₂ levelsbut measures the apparent ‘total’ Aβ₄₂. There was a dose andconcentration dependent increase in Aβ₄₂ (with plateau levels ofapproximately 300, 125 and 25 pg/ml detected following 10, 1 or 0.1mg/kg H2L1 respectively). From the analysis, the increase in the “totalAβ₄₂” is likely to be due to the result of a significant efflux ofamyloid from outside the plasma pool, that appeared dependent upon H2L1concentrations >1 ug/mL, and did not appear to be a result of lack ofclearance of complex. This was evident by the elimination rate of thetotal Aβ₄₂ as well as the fluctuation in the total levels over a dosinginterval.

To date only the plasma analysis has been completed and fully analysed.However preliminary analysis indicates that there was a trend towardsreduction in CSF and increase in the hippocampal level of ‘total’ Aβ42(measured as generally described above) following treatment with 10mg/kg H2L1.

In some brain sections, small areas of microhaemorrhage were detected asshown by the Perls' Prussian Blue staining method. This methodvisualises ferric iron (iron is an essential constituent of theoxygen-carrying haemoglobin found in red cells) by producing aninsoluble blue compound. However there was no difference in theincidence between vehicle and drug treated animals.

Analyses on Aged Cynomologus Macaque Monkeys for Beta Amyloid Plaques inthe Brain and Total Beta Amyloid in the Plasma

Cerebral spinal fluid (CSF) and tissue parameters of human AD have beendisplayed in the cynomolgus monkey. The aged cynomolgus monkey has beenshown to have evidence of amyloid deposition. (Covance, The cynomolgusmonkey as a model for Alzheimer's disease. In: Buse E, Habermann G,Friderichs-Gromoll S, Kaspereit J, Nowak P and Niggemann K, editors.Poster Presentation at the 44th Annual Meeting of the Society ofToxicology, New Orleans, La., 6 to 10 Mar. 2005). The potential for H2L1to elicit an inappropriate response (such as encephalitis) in an agedbrain was investigated in old, ca. 20 years, ex-breeding female monkeys.In addition, safety, treatment-related microhaemorrhage,neutralization/clearance of test material, hypersensitivity, and immunecomplex disease were also investigated following intravenousadministration for 8 weeks in two-weekly intervals. In addition CNS andblood samples were analysed for levels of Aβ_(40/42).

Study Design

Groups of 5 (group 1), 9 (group 2) or 10 (group 3) geriatric femalecynomolgus monkeys were given 0 (vehicle), 50 or 100 mg/kg/dosing dayH2L1 in vehicle (4 ml/kg) every second week for 8 weeks intravenously byslow bolus administration. The vehicle consisted of sodium acetatetrihydrate 6.81 mg/mL, disodium edetate dehydrate 0.0186 mg/mL,polysorbate 80 0.2 mg/mL, and L-Arginine base 10 mg/mL, the pH was 5.5.Dose levels were chosen to investigate dose levels that were 5 and 10fold intended clinical dose levels.

The following evaluations were performed pre-dose, daily (clinicalsigns, body weight, food consumption), week 4 and the week beforenecropsy: in-life animal observations, body weight, body temperature,haematology, clinical chemistry (including cerebrospinal fluid [CSF]analysis), urinalysis, and cytokine determination in CSF. Followingnecropsy, organ weights, macroscopic observations, and microscopicobservations of the brain, cervical spinal cord and gross lesions wereconducted on all animals. Toxicokinetic evaluation was performed aftereach dosing.

Results

There were no unscheduled deaths, and there were no signs whichindicated an influence of the test item on the general condition of theanimals at the administered doses. The only remarkable observations inclinical pathology (hematology and serum chemistry) were concluded to beage- and not test article related.

Systemic exposure to H2L1 (as measured by AUC_(0-τ) and C_(max))increased approximately in proportion to dose. For both dose groups,there was no marked change (≧2-fold) in systemic exposure between the1st dose and 4th dose sampling periods.

There were no signs of inflammatory reactions in the brain detected byCSF-analysis, and there were no macroscopic or microscopic findings atnecropsy that suggested a test item influence, specifically nomicrohaemorrhage or encephalitis.

This study was conducted in compliance with the Good Laboratory PracticeRegulations as outlined in German Chemical Law, annex 1 and 2 to §19aChemikalien Gesetz, June 2002, the OECD Principles of Good LaboratoryPractice (revised 1997, issued January 1998) ENV/MC/CHEM (98) 17, theConsensus Document “The Application of the OECD Principles of GLP to theOrganisation and Management of Multi-Site Studies” ENV/JM/MONO(2002)9.Studies conducted in compliance with the above regulations and standardswere considered acceptable to US FDA regulatory authorities.

Analysis of Plague Load in the CNS

The left brain hemispheres of the vehicle treated cynomologus macaquemonkeys from the above study were analysed by immunohistochemistry. Acoronal section, at the level of the middle temporal sulcus containingportions of the dentate gyrus and hippocampus, was processed into wax asdescribed above. For immunohistochemistry, sections were labelled with apan-Aβ antibody (1E8, monoclonal antibody raised to Aβ 13-27), or withthe Aβ42 antibody, (20G10, monoclonal antibody recognising Aβ x-42), andlabelling was developed as above. A visual count of the number ofplaques was taken for each section. Tissue from all five vehicle-treatedcynomolgus monkeys showed evidence of parenchymal Aβ plaques. There wasalso evidence of cerebrovascular labelled Aβ and intraneuronal Aβ.

Analysis of Beta Amyloid/Antibody Complexes in Plasma

Biochemical analysis was carried out on plasma samples from two timepoints (at the end of weeks 4 and 8 after start of dosing) from animalsdosed with 50 mg/kg (n=9) or 100 mg/kg (n=10) H2L1, or vehicle dosedcontrols (n=5). 100 ul duplicate samples were analysed using thecommercially available Innogenetics Aβ 1-42 ELISA kit, incubatedovernight at 4° C. Control samples were analysed both neat and at 1:10dilution (using the supplied diluent), while samples from the dosedanimals were tested neat and at 1:25. Subsequent absorbance values wereanalysed, with unknown absorbance values backcalculated to pg/ml valuesusing standard curves, and then corrected for any assay dilution. Totalplasma levels of Aβ42 derived from these samples are shown in Table 12below (figures in pg/ml±SE); all samples from animals treated with H2L1contained significantly higher levels of Aβ42 (p<0.001 by studentt-test) than in control groups.

TABLE 12 Week 4 (pg/ml) Week 8 (pg/ml) Control (1:10) 104.1 ± 30.4 29.8± 7.9 50 mg/kg (1:25) 830.5 ± 79.1 615.8 ± 50.2 100 mg/kg (1:25) 1020.5± 84.4  492.7 ± 46.3

Data reported were obtained from diluted samples. Results from the neatsamples were not used as many data points were either greater than thetop standard, or due to sample volume limitation, only assayed as asingle point.

Production Process

Expression vectors encoding H2L1 and operatively linked to amplifiableselection markers such as the DHFR or glutamine synthetase may be usedto transfect or transduce appropriate parental CHO cell lines (egCHODG44 or CHOK 1) to produce engineered cell lines suitable forproduction of monoclonal antibody at large scale (for review seeBebbington and Hentschel DNA Cloning Volume III; A practical approach(edited by Glover DM) (Oxford IRL press, 1987). In order to increaseexpression levels the coding sequence maybe codon optimized in order toavoid cis-acting sequence motifs and extreme GC contents (high or low).SEQ. ID Nos:42 and No:43 exemplify such a coding sequence for H2 heavychain and L1 light chain. Large scale production maybe in stirred tankbioreactors using animal-derived-component-free medium, followed bypurification. This may comprise clarification of the harvest, followedby Protein-A affinity chromatography, and further purification using ion(e.g. cation) exchange and mixed mode (e.g. ceramic hydroxyapatite)chromatography unit operations. A virus removal nanofiltration isfollowed by a final ultrafiltration/diafiltration step that enablesformulation suitable for the intended route of administration.

Example of Pharmaceutical Formulation

Ingredient Quantity (per mL) H2L1 50 mg Sodium acetate trihydrate 6.81mg Polysorbate 80 0.20 mg Arginine base 10.00 mg Sodium chloride 3.00 mgDisodium edetate dihydrate 0.0186 mg Hydrochloric acid qs to give pH 5.5Water for Injections To make 1.0 mL Nitrogen To fill headspace

Amino Acid Sequences of V-Regions of Acceptor Frameworks and HumanisedVariants

M99675 heavy chain acceptor framework V region, amino acid sequence(SEQ ID No: 21) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARM99675 heavy chain acceptor framework V region DNA (SEQ ID No: 22)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTAGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GACAA51135 light chain acceptor framework V region amino acid sequence(SEQ ID No: 24) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQ TPWTFGQGTKVEIKCAA51135 light chain acceptor framework V region DNA (SEQ ID No: 25)GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAHumanised heavy chain V region variant H1 , amino acid sequence(SEQ ID No: 26) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNGMAWVRQAPGKGLEWVSFISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVS GTWFAYWGQGTLVTVSSHumanised heavy chain V region variant H1 DNA  coding sequence(SEQ ID No: 27) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACAACGGAATGGCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTCAGCGGGACCTGGTTTGCTTACTGGGGCCAGGGCACACTAGTCACAGTCTCCT CAHumanised heavy chain V region variant H2, amino acid sequence(SEQ ID No: 28) EVQLVESGGGLVQPGGSLRLSCAVSGFTFSDNGMAWVRQAPGKGLEWVSFISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVS GTWFAYWGQGTLVTVSSHumanised heavy chain V region variant H2 DNA (SEQ ID No: 29)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTCAGTGACAACGGAATGGCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTCAGCGGGACCTGGTTTGCTTACTGGGGCCAGGGCACACTAGTCACAGTCTCCT CAHumanised heavy chain V region variant H3, amino acid sequence(SEQ ID No: 30) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNGMAWVRQAPGKGLEWISFISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVS GTWFAYWGQGTLVTVSSHumanised heavy chain V region variant H3 DNA (SEQ ID No: 31)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACAACGGAATGGCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCTCATTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTCAGCGGGACCTGGTTTGCTTACTGGGGCCAGGGCACACTAGTCACAGTCTCCT CAHumanised light chain V region variant L1 amino acid sequence(SEQ ID No: 32) DIVMTQSPLSLPVTPGEPASISCRVSQSLLHSNGYTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTRH VPYTFGGGTKVEIKHumanised light chain V region variant L1 DNA (SEQ ID No: 33)GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGAGTTAGTCAGAGCCTTTTACACAGTAATGGATACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCAACCGATTTTCTGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCTCTCAAACTAGACATGTTCCGTACACGTTCGGCGGAGGGACCAAGGTGGAAATCAAAMature H1 heavy chain amino acid sequence(Fc mutated double mutation bold) (SEQ ID No: 34)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNGMAWVRQAPGKGLEWVSFISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGTWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKH1 Full length DNA (SEQ ID No: 35)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACAACGGAATGGCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTCAGCGGGACCTGGTTTGCTTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAAMature H2 heavy chain amino acid sequence,(Fc mutated double mutation bold) (SEQ ID No: 36)EVQLVESGGGLVQPGGSLRLSCAVSGFTFSDNGMAWVRQAPGKGLEWVSFISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGTWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKH2 Full length DNA (SEQ ID No: 37)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGTCTCTGGATTCACCTTCAGTGACAACGGAATGGCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTCAGCGGGACCTGGTTTGCTTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAAMature H3 heavy chain amino acid sequence(Fc mutated double mutation bold) (SEQ ID No: 38)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNGMAWVRQAPGKGLEWISFISNLAYSIDYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSGTWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKH3 full length DNA (SEQ ID No: 39)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACAACGGAATGGCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCTCATTCATTAGTAATTTGGCATATAGTATCGACTACGCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTCAGCGGGACCTGGTTTGCTTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAAMature Light chain amino acid sequence (SEQ ID No: 40)DIVMTQSPLSLPVTPGEPASISCRVSQSLLHSNGYTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTRHVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC L1 Full length DNA (SEQ ID No: 41)GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGAGTTAGTCAGAGCCTTTTACACAGTAATGGATACACCTATTTACATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATAAAGTTTCCAACCGATTTTCTGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCTCTCAAACTAGACATGTTCCGTACACGTTCGGCGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA GCTTCAACAGGGGAGAGTGTOptimised H2 heavy chain DNA (SEQ ID No: 42)GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGAGCTGTGCCGTGTCCGGCTTCACCTTCAGCGACAACGGCATGGCCTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCTTCATCAGCAACCTGGCCTACAGCATCGACTACGCCGACACCGTGACCGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTGAGCGGCACCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGGCCGGAGCCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTG TCCCCTGGCAAGOptimised L1 light chain DNA (SEQ ID No: 43)GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCTGGCGAGCCCGCCAGCATCAGCTGTAGAGTGAGCCAGAGCCTGCTGCACAGCAACGGCTACACCTACCTGCACTGGTATCTGCAGAAGCCTGGCCAGAGCCCTCAGCTGCTGATCTACAAGGTGTCCAACCGGTTCAGCGGCGTGCCTGATAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGATGTGGGCGTGTACTACTGCTCCCAGACCAGACACGTGCCTTACACCTTTGGCGGCGGAACAAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA GCTTCAACCGGGGCGAGTGC

1. A method of treating a β-amyloid peptide related disease in a humanpatient comprising administering to the patient an effective amount ofan isolated antibody or antigen binding fragment thereof which bindsb-amyloid peptide and which comprises the following CDRs: CDRH1:(SEQ ID No: 1) DNGMA CDRH2: (SEQ ID No: 2) FISNLAYSIDYADTVTG CDRH3:(SEQ ID No :3) GTWFAY

within a human heavy chain variable region originating from the VH3 genefamily and: CDRL1: (SEQ ID No: 4) RVSQSLLHSNGYTYLH CDRL2: (SEQ ID No: 5)KVSNRFS CDRL3: (SEQ ID No: 6) SQTRHVPYT

within a human light chain variable region originating from the aminoacid sequence disclosed in GenPept entry CAA51135 (SEQ ID No:24).
 2. Themethod of claim 1 wherein an isolated antibody has the human heavy chainvariable region which originates from a V gene selected from the groupconsisting of: VH3-48, VH3-21, VH3-11, VH3-7, VH3-13, VH3-74, VH3-64,VH3-23, VH3-38, VH3-53, VH3-66, VH3-20, VH3-9 and VH3-43.
 3. The methodof claim 2 wherein an isolated antibody has a human acceptor heavy chainframework of M99675 (SEQ ID No:21) together with a framework
 4. 4. Themethod of claim 3 wherein an isolated antibody has the framework 4sequence encoded by the human JH4 minigene (Kabat): (SEQ ID No: 23)YFDYWGQGTLVTVSS

of which the initial four residues fall within the CDR3 region isreplaced by the incoming CDR from a donor antibody.
 5. The method ofclaim 1 wherein an isolated antibody contains one or more substitutionsof amino acid residues based on the corresponding residues found in adonor V_(H) domain having the sequence: SEQ ID No:17 and V_(L) domainhaving the sequence: SEQ ID No: 19 that maintain all or substantiallyall of the binding affinity of the donor antibody for b-amyloid peptide.6. The method of claim 5 wherein an isolated antibody has a humanacceptor heavy chain framework of M99675 together with JH4 containingone to four amino acid residue substitutions selected from positions 24,48, 93 and/or 94 (Kabat numbering).
 7. The method of claim 6 wherein anisolated antibody has a human acceptor heavy chain framework whichcomprises the following residues: Position Residue (i) 93 V 94 S or (ii)24 V 93 V 94 S or (iii) 48 I 93 V 94 S


8. The method of claim 1 wherein an isolated antibody binds b-amyloidpeptide comprising a V_(H) chain having the sequence set forth in SEQ IDNo:26 and a V_(L) domain having the sequence set forth in SEQ ID No:32.9. The method of claim 1 wherein an isolated therapeutic antibody bindsb-amyloid peptide comprising a V_(H) chain having the sequence set forthin SEQ ID No: 28 and a V_(L) domain having the sequence set forth in SEQID No:32.
 10. The method of claim 1 wherein an isolated therapeuticantibody binds b-amyloid peptide comprising a V_(H) chain having thesequence set forth in SEQ ID No:30 and a V_(L) domain having thesequence set forth in SEQ ID No:32.
 11. The method of claim 1 wherein anisolated antibody or antigen binding fragment thereof binds b-amyloidpeptide 1-12 (SEQ ID No:15) with equilibrium constant KD less than 100μM and has an equilibrium constant KD for binding to b-amyloid peptide2-13 (SEQ ID No:44) which is 1000-fold greater than that for peptide1-12 (SEQ ID No:15), both determinations being made in a surface plasmonresonance assay utilising peptide captured on streptavidin chip.
 12. Themethod of claim 1 wherein an isolated therapeutic antibody or antigenbinding fragment thereof binds b-amyloid peptide 1-40 with equilibriumconstant KD less than 10 nM and has an equilibrium constant KD forbinding to b-amyloid peptide 2-13 (SEQ ID No:44) which is 1000-foldgreater than that for peptide 1-12 (SEQ ID No:15), both determinationsbeing made in the surface plasmon resonance assay described in Method Bof the Examples.
 13. The method of claim 1 wherein an isolatedtherapeutic antibody according claim 1 is of IgG1 isotype.
 14. Themethod of claim 1 wherein an isolated antibody essentially lacks thefunctions of an activation of complement by the classical pathway; andb) mediating antibody-dependent cellular cytotoxicity.
 15. The method ofclaim 13 wherein an isolated antibody in which residues 235 and 237 havebeen mutated to alanine.
 16. The method of claim 1 wherein an isolatedantibody comprises a heavy chain having the sequence set forth in SEQ IDNo:34, 36 or 38 and a light chain having the sequence set forth in SEQID No:40.
 17. The method of claim 1 wherein an isolated antibody or afragment thereof binds b-amyloid peptide comprising a V_(H) domainhaving the sequence: SEQ ID No:17 and a V_(L) domain having thesequence: SEQ ID No:19.